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1.
Anticancer Res ; 39(10): 5369-5374, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570431

RESUMO

BACKGROUND/AIM: Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. MATERIALS AND METHODS: The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA. RESULTS: P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. CONCLUSION: P60 may potentiate CIK cell cytotoxicity against tumor cells.


Assuntos
Células Matadoras Induzidas por Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Fatores de Transcrição Forkhead/antagonistas & inibidores , Neoplasias Renais/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Peptídeos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura/métodos , Células Matadoras Induzidas por Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Neoplasias Renais/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo
2.
Bioresour Technol ; 293: 122062, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31494436

RESUMO

The study aimed to evaluate biopolymer production using two bacterial strains, Acinetobacter junii BP25 and Aeromonas hydrophila ATCC 7966, and their co-culture. Batch experiments were evaluated using acetate and butyrate as carbon sources in feast and famine strategy. Feast phase was studied using carbon, nitrates and phosphate in the ratio of 100:8:1 and famine phase was limited with the phosphate and nitrates. Co-culture resulted in highest specific growth rate (0.30 h-1) in the feast phase and the famine phase accounted the maximum polyhydroxybutyrate (PHB) accumulation (2.46 g PHB/L), followed by Acinetobacter junii BP25 (0.25 h-1 and 1.82 g PHB/L) and Aeromonas hydrophila ATCC 7966 (0.17 h-1 and 1.12 g PHB/L). Fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (NMR) structural analysis confirmed as PHB. PHB production using the co-culture could be integrated with biohydrogen process using volatile fatty acids (VFA) as a carbon source in the biorefinery framework.


Assuntos
Acinetobacter , Butiratos , Aeromonas hydrophila , Reatores Biológicos , Técnicas de Cocultura , Hidroxibutiratos , Poliésteres , Espectroscopia de Infravermelho com Transformada de Fourier
3.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 33(10): 1310-1319, 2019 Oct 15.
Artigo em Chinês | MEDLINE | ID: mdl-31544445

RESUMO

Objective: To investigate the heterotopic osteogenesis of tissue engineered bone using the co-culture system of vascular endothelial cells (VECs) and adipose-derived stem cells (ADSCs) as seed cells. Methods: The partially deproteinized biological bone (PDPBB) was prepared by fibronectin combined with partially deproteinized bone (PDPB). The ADSCs of 18-week-old Sprague Dawley (SD) rats and VECs of cord blood of full-term pregnant SD rats were isolated and cultured. Three kinds of tissue engineered bone were constructed in vitro: PDPBB+VECs (group A), PDPBB+ADSCs (group B), PDPBB+co-cultured cells (VECs∶ADSCs was 1∶1, group C), and PDPBB was used as control group (group D). Scanning electron microscopy was performed at 10 days after cell transplantation to observe cell adhesion on scaffolds. Forty-eight 18-week-old SD rats were randomly divided into groups A, B, C, and D, with 12 rats in each group. Four kinds of scaffolds, A, B, C, and D, were implanted into the femoral muscle bags of rats in corresponding groups. The animals were killed at 2, 4, 8, and 12 weeks after operation for gross observation, HE staining and Masson staining histological observation, and the amount of bone collagen was measured quantitatively by Masson staining section. Results: Scanning electron microscopy showed that the pores were interconnected in PDPB materials, and a large number of lamellar protein crystals on the surface of PDPBB modified by fibronection were loosely attached to the surface of the scaffold. After 10 days of co-culture PDPBB and cells, a large number of cells attached to PDPBB and piled up with each other to form cell clusters in group C. Polygonal cells and spindle cells were mixed and distributed, and some cells grew along bone trabeculae to form cell layers. Gross observation showed that the granulation tissue began to grow into the material pore at 2 weeks after operation. In group C, a large number of white cartilage-like substances were gradually produced on the surface of the material after 4 weeks, and the surface of the material was uneven. At 12 weeks, the amount of blood vessels on the surface of group A increased, and the material showed consolidation; there was a little white cartilage-like material on the surface of group B, but the pore size of the material did not decrease significantly; in group D, the pore size of the material did not decrease significantly. Histological observation showed that there was no significant difference in the amount of bone collagen between groups at 2 weeks after operation ( F=2.551, P=0.088); at 4, 8, and 12 weeks after operation, the amount of bone collagen in group C was significantly higher than that in other 3 groups, and that in group B was higher than that in group D ( P<0.05); there was no significant difference between group A and groups B, D ( P>0.05). Conclusion: The ability of heterotopic osteogenesis of tissue engineered bone constructed by co-culture VECs and ADSCs was the strongest.


Assuntos
Células Endoteliais , Osteogênese , Animais , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Mesenquimais , Ratos , Ratos Sprague-Dawley , Células-Tronco , Engenharia Tecidual , Tecidos Suporte
4.
Toxicol Lett ; 316: 10-19, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31476341

RESUMO

Rapid risk assessment models for different types of cigarette smoke extract (CSE) exposure are critical to understanding the etiology of chronic obstructive pulmonary disease. The present study investigated inflammation of cultured tracheal tissues with CSE exposure. Rat trachea rings were isolated, cultured, then exposed to various concentrations of CSE from 3R4 F reference cigarettes for 4 h. Tissue/cellular morphology, ultrastructure, viability and damage, inflammatory cell infiltration, and inflammatory protein levels were measured and compared to untreated controls. Human bronchial epithelial cells (BEAS-2B) exposed to 0 or 300 µg/mL CSE were cocultured with macrophages to assess extent of mobilization and phagocytosis. Endotracheal epithelium cilia densities were significantly reduced with increasing CSE concentrations, while mucous membranes became increasingly disordered; both eventually disappeared. Macrophages became larger as the CSE concentration increased, with microvilli and extended pseudopodium covering their surface, and many primary and secondary lysosomes present in the cytoplasm. Inflammatory cell infiltration also increased with increasing CSE dose, as did intracellular adhesion molecule-1(ICAM-1), interleukin-6(IL-6). The method described here may be useful to qualitatively characterized the effects of the compound under study. Then, we use BEAS-2B cell line system to strength the observation made in the cultured tissues. Probably, an approach to integrate results from both experiments will facilitate its application. These results demonstrate that cultured rat tracheal rings have a whole-tissue structure that undergoes inflammatory processes similar to in vivo tissues upon CSE exposure.


Assuntos
Células Epiteliais/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/etiologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Tabaco/efeitos adversos , Traqueia/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Humanos , Mediadores da Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Masculino , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Ratos Sprague-Dawley , Medição de Risco , Fatores de Tempo , Técnicas de Cultura de Tecidos , Traqueia/metabolismo , Traqueia/ultraestrutura
5.
Toxicol Lett ; 316: 73-84, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31513886

RESUMO

In the liver microenvironment, interactions among diverse types of hepatic cells are involved in liver fibrosis. In fibrotic tissues, exosomes act as transporters in intercellular communication. Long non-coding RNAs (lncRNAs) are involved in the activation of hepatic stellate cells (HSCs), which are participants in liver fibrosis. However, the functions of exosomal lncRNAs in liver fibrosis induced by arsenite are undefined. The purposes of the present study were (a) to determine if lncRNAs secreted from human hepatic (L-02) cells exposed to arsenite are shuttled to hepatic stellate LX-2 cells and (b) to establish their effects on LX-2 cells. In mice, MALAT1 was overexpressed in the progression of liver fibrosis induced by arsenite as well as in L-02 cells exposed to arsenite. Co-cultures with arsenite-treated L-02 cells induced the activation of LX-2 cells and overexpression of MALAT1. Arsenite-treated L-02 cells transported MALAT1 into LX-2 cells. Downregulation of MALAT1, which reduced the MALAT1 levels in exosomes derived from arsenite-treated L-02 cells, inhibited the activation of LX-2 cells. Additionally, exosomal MALAT1 derived from arsenite-treated L-02 cells promoted the activation of LX-2 cells via microRNA-26b regulation of COL1A2. Furthermore, circulating exosomal MALAT1 was up-regulated in people exposed to arsenite. In sum, exosomes derived from arsenite-treated hepatic cells transferred MALAT1 to HSCs, which induced their activation. These findings support the concept that, during liver fibrosis induced by arsenite, exosomal lncRNAs are involved in cell-cell communication.


Assuntos
Arsenitos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Exossomos/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática Experimental/metabolismo , Fígado/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Compostos de Sódio , Animais , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Técnicas de Cocultura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Exossomos/genética , Exossomos/ultraestrutura , Regulação da Expressão Gênica , Células Estreladas do Fígado/ultraestrutura , Humanos , Fígado/ultraestrutura , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Masculino , Camundongos , MicroRNAs/genética , RNA Longo não Codificante/genética , Transdução de Sinais
6.
J Microbiol ; 57(9): 738-747, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31376106

RESUMO

The simultaneous removal of phenol and selenite from synthetic wastewater was investigated by adopting two different co-culturing techniques using the fungus Phanerochaete chrysosporium and the bacterium Delftia lacustris. Separately grown biomass of the fungus and the bacterium (suspended co-culture) was incubated with different concentrations of phenol (0-1,200 mg/L) and selenite (10 mg/L). The selenite ions were biologically reduced to extracellular Se(0) nanoparticles (3.58 nm diameter) with the simultaneous degradation of up to 800 mg/L of phenol. Upon growing the fungus and the bacterium together using an attached growth co-culture, the bacterium grew as a biofilm onto the fungus. The extracellularly produced Se(0) in the attached growth co-culture had a minimum diameter of 58.5 nm. This co-culture was able to degrade completely 50 mg/L phenol, but was completely inhibited at a phenol concentration of 200 mg/L.


Assuntos
Delftia/metabolismo , Phanerochaete/metabolismo , Fenol/metabolismo , Ácido Selenioso/metabolismo , Selênio/metabolismo , Biodegradação Ambiental , Biotransformação , Técnicas de Cocultura , Delftia/crescimento & desenvolvimento , Oxirredução , Phanerochaete/crescimento & desenvolvimento , Águas Residuárias/microbiologia , Poluentes da Água/metabolismo
7.
World J Microbiol Biotechnol ; 35(9): 137, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432268

RESUMO

The presence of very high concentrations of organic pollutants, phenols, tannins and heavy metals mainly chromium in wastewater discharged from leather industries, tags it as one of the most polluting industries. The phenolic syntans discharged from tanning units have an adverse effect on living organisms and cause serious environmental pollution, thereby making it very imperative to remove it. Among various treatment methods available for removal of phenols, biodegradation is environment friendly. The present study aims at the remediation of phenolic syntan used in the leather industry employing individual as well as co-culture of Bacillus cereus and Pseudomonas aeruginosa at varying syntan concentration in the medium. Parameters such as chemical oxygen demand (COD), total organic carbon (TOC), total phenol content (TPC) and Fourier Transform Infrared Spectroscopy (FTIR) indicating biodegradation were analyzed. Promising results were observed with P. aeruginosa, which exhibited a reduction in TPC by 62-72% in all the concentrations of syntan tested just within 12 h of inoculation, whereas about 67 and 83% reduction in COD and TOC respectively was observed for 2000 ppm concentration at the end of 5 days. B. cereus also demonstrated very good reduction in the above parameters however; percentage was less as compared to P. aeruginosa. In the case of co-culture, the TPC reduction was higher than B. cereus but lesser than P. aeruginosa. The percentage reduction in TOC and COD was highest for 500 ppm which eventually decreased for subsequent concentrations.


Assuntos
Bacillus cereus/metabolismo , Resíduos Industriais , Fenóis/metabolismo , Pseudomonas aeruginosa/metabolismo , Poluentes Químicos da Água/metabolismo , Bacillus cereus/crescimento & desenvolvimento , Análise da Demanda Biológica de Oxigênio , Biotransformação , Carbono/análise , Técnicas de Cocultura , Fenóis/análise , Pseudomonas aeruginosa/crescimento & desenvolvimento , Espectroscopia de Infravermelho com Transformada de Fourier , Água/química
9.
Cancer Sci ; 110(10): 3079-3088, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432594

RESUMO

Chimeric antigen receptor-engineered T (CAR-T)-cell therapy holds significant promise for the treatment of hematological malignancies, especially for B-cell leukemia and lymphoma. However, its efficacy against non-hematological malignancies has been limited as a result of several biological problems characteristic of the tumor microenvironment of solid tumors. One of the main hurdles is the heterogeneous nature of tumor-associated antigens (TAA) expressed in solid tumors. Another hurdle is the inefficient activation and limited persistence of CAR-T cells, mainly as a result of T-cell exhaustion caused by immunosuppressive factors in the tumor microenvironment. In the present study, to address these problems, we engineered CAR-T cells to produce antagonistic anti-programmed cell death protein 1 (PD-1) single-chain variable fragment (scFv), by which PD-1-dependent inhibitory signals in CAR-T cells and adjacent tumor-specific non-CAR-T cells are attenuated. In mouse solid tumor models, PD-1 scFv-producing CAR-T cells induced potent therapeutic effects superior to those of conventional CAR-T cells, along with a significant reduction of apoptotic cell death not only in CAR-T cells themselves but also in TAA-specific T cells in the tumor tissue. In addition, the treatment with anti-PD-1 scFv-producing CAR-T cells resulted in an increased concentration of PD-1 scFv in tumor tissue but not in sera, suggesting an induction of less severe systemic immune-related adverse events. Hence, the present study developed anti-PD-1 scFv-producing CAR-T cell technology and explored its cellular mechanisms underlying potent antitumor efficacy.


Assuntos
Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptor de Morte Celular Programada 1/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Técnicas de Cocultura , Masculino , Camundongos , Neoplasias/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Microbiol Res ; 228: 126305, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422234

RESUMO

Traditional culture-based enumeration methods were compared with the ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) technique to assess Bdellovibrio-and-like-organisms (BALOs) predator-prey interactions. Gram-negative [Pseudomonas spp. and Klebsiella pneumoniae (K. pneumoniae)] and Gram-positive [Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium)] organisms were employed as prey cells, while a Bdellovibrio bacteriovorus strain (PF13) was used as the predator. The co-culture experiments were also compared in diluted nutrient broth (DNB) and HEPES buffer. In both media, K. pneumoniae (maximum log reduction of 5.13) and Pseudomonas fluorescens (P. fluorescens) (maximum log reduction of 4.21) were sensitive to predation by B. bacteriovorus PF13 as their cell counts and gene copies were reduced during all the co-culture experiments, while the concentration of B. bacteriovorus PF13 increased. The concentration of B. bacteriovorus PF13 also increased in the presence of S. aureus (HEPES buffer) and E. faecium (DNB), indicating that the predator interacted with these Gram-positive prey in order to survive. Moreover, as no predator plaques were produced in the co-culture experiments with P. aeruginosa (DNB and HEPES buffer), S. aureus (DNB and HEPES buffer) and E. faecium (HEPES buffer), EMA-qPCR proved to be beneficial in monitoring the concentration of B. bacteriovorus. In conclusion, the cell counts and/or EMA-qPCR analysis for the HEPES buffer and DNB assays were successfully employed to monitor the predation of P. fluorescens and K. pneumoniae by B. bacteriovorus, while E. faecium was sensitive to predation in DNB and S. aureus was sensitive to predation in HEPES buffer.


Assuntos
Azidas , Fenômenos Fisiológicos Bacterianos , Interações Microbianas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Bactérias , Bdellovibrio bacteriovorus/fisiologia , Técnicas de Cocultura , Contagem de Colônia Microbiana , Enterococcus faecium/fisiologia , Klebsiella pneumoniae/fisiologia , Pseudomonas/fisiologia , Staphylococcus aureus/fisiologia , Águas Residuárias/microbiologia , Purificação da Água
11.
Microbiol Res ; 227: 126292, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421719

RESUMO

Azotobacter chroococcum (Az) and Trichoderma viride (Tv) represent agriculturally important and beneficial plant growth promoting options which contribute towards nutrient management and biocontrol, respectively. When Az and Tv are co-cultured, they form a biofilm, which has proved promising as an inoculant in several crops; however, the basic aspects related to regulation of biofilm formation were not investigated. Therefore, whole transcriptome sequencing (Illumina NextSeq500) and gene expression analyses were undertaken, related to biofilm formation vis a vis Tv and Az growing individually. Significant changes in the transcriptome profiles of biofilm were recorded and validated through qPCR analyses. In-depth evaluation also identified several genes (phoA, phoB, glgP, alg8, sipW, purB, pssA, fadD) specifically involved in biofilm formation in Az, Tv and Tv-Az. Genes coding for RNA-dependent RNA polymerase, ABC transporters, translation elongation factor EF-1, molecular chaperones and double homeobox 4 were either up-regulated or down-regulated during biofilm formation. To our knowledge, this is the first report on the modulation of gene expression in an agriculturally beneficial association, as a biofilm. Our results provide insights into the regulatory factors involved during biofilm formation, which can help to improve the beneficial effects and develop more effective and promising plant- microbe associations.


Assuntos
Azotobacter/genética , Biofilmes/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Interações Microbianas/genética , Transcriptoma , Trichoderma/genética , Técnicas de Cocultura , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Bacterianos/genética , Genes Fúngicos/genética , Interações Microbianas/fisiologia , Desenvolvimento Vegetal , Plantas/microbiologia , Regulação para Cima
12.
Sheng Li Xue Bao ; 71(4): 575-580, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31440754

RESUMO

The aim of the present study was to investigate the effect of salidroside (Sal) on inflammatory activation induced by lipopolysaccharide (LPS) in the co-culture of rat alveolar macrophages (AM) NR 8383 and type II alveolar epithelial cells (AEC II) RLE-6TN. CCK-8 colorimetric method was used to detect cell proliferation percentage. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-10 (IL-10) in the supernatant. Western blot was used to examine the expression levels of phosphorylated AKT (p-AKT) and total AKT protein. The results showed that pretreatment of RLE-6TN cells or co-culture of RLE-6TN and NR 8383 cells with 32 and 128 µg/mL Sal for 1 h, followed by continuous culture for 24 h, significantly increased the cell proliferation (P < 0.05). Compared with control group, 32 and 128 µg/mL Sal pretreatment significantly increased the ratio of p-AKT/AKT in RLE-6TN cells (P < 0.05). Pretreatment of 32 µg/mL Sal not only inhibited the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05), but also enhanced the inhibitory effect of RLE-6TN and NR 8383 cells co-culture on the secretion of TNF-α and MIP-2 by NR 8383 cells induced by LPS (P < 0.05). In addition, 32 µg/mL Sal pretreatment promoted LPS-induced IL-10 secretion by NR 8383 cells (P < 0.05), and enhanced the promoting effect of co-culture of RLE-6TN and NR 8383 cells on the IL-10 secretion by LPS-induced NR 8383 cells (P < 0.05). In conclusion, Sal may directly inhibit LPS-induced inflammatory activation of AM (NR 8383), promote the proliferation of AEC II (RLE-6TN) through PI3K/AKT signaling pathway, and enhance the regulatory effect of AEC II on LPS-induced inflammatory activation of AM.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Glucosídeos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Fenóis/farmacologia , Transdução de Sinais , Células Epiteliais Alveolares/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL2/metabolismo , Técnicas de Cocultura , Interleucina-10/metabolismo , Lipopolissacarídeos , Macrófagos Alveolares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismo
13.
Life Sci ; 233: 116714, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31376370

RESUMO

Increased levels of particulate matter (PM) air pollutants in East Asia have resulted in detrimental health impacts increasing morbidity and mortality. Epidemiological studies suggest a possible relation between the cutaneous exposure of PM and increased oxidative stress and inflammation which lead to skin lesions. The present study utilizes an integrated cell culture model of keratinocytes and fibroblasts to mimic viable skin layers and investigate the possible effects of PM exposure after penetration through corneocytes. The skin perfection is upheld by homeostatic functionality of epidermal cells and the integrity of connective tissues. Exposure to xenobiotics could alter the skin cell homeostasis aggravating premature skin aging. Stimulation of HaCaT keratinocytes by PM collected from Beijing, China (CPM) increased the intracellular ROS levels triggering a cascade of events aggravating inflammatory responses and connective tissue degradation. In HDF fibroblasts, treatment with preconditioned keratinocyte culture media augmented inflammatory responses, cellular differentiation, and connective tissue degradation. Above events were marked by the increased intracellular ROS, inflammatory mediators, pro-inflammatory cytokines, matrix metalloproteinases (MMP)-1 and -2 levels, collagenase, and elastase activity. Fucosterol treatment of keratinocytes dose-dependently attenuated the detrimental effects both in keratinocytes and fibroblasts restoring the conditions near to physiological levels. Further evaluations could be advanced on developing fucosterol, in forms such as rejuvenating cosmeceuticals which could attenuate detrimental responses of CPM exposure.


Assuntos
Fibroblastos/efeitos dos fármacos , Inflamação/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Material Particulado/efeitos adversos , Dermatopatias/tratamento farmacológico , Pele/efeitos dos fármacos , Estigmasterol/análogos & derivados , Poluentes Atmosféricos/efeitos adversos , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , NF-kappa B/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Dermatopatias/etiologia , Dermatopatias/metabolismo , Dermatopatias/patologia , Estigmasterol/farmacologia
14.
Cancer Immunol Immunother ; 68(9): 1455-1465, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31444606

RESUMO

Macrophages have been shown to infiltrate a wide range of malignancies and are often considered to promote tumour survival, growth and spread. However, the source and behaviour of discrete tumour-associated macrophage populations are still poorly understood. Here we show a novel method for the rational development of bone marrow-derived monocytes appropriate for the study of processes which involve the contribution of circulating inflammatory monocytes. We have shown that in response to tumour-conditioned medium, these cells upregulate CD206 and CD115, markers traditionally associated with M2-type macrophages. Treated cells show reduced capacity for cytokine secretion but significantly impact CD4+ and CD8+ T-cell proliferation and polarization. Coculture with conditioned bone marrow-derived monocytes significantly reduced CD4+ T-cell proliferation but increased CD8+ T-cell proliferation and granzyme B expression with significant induction of IFNγ secretion by both CD4+ and CD8+ T cells, indicating that these cells may have a role in promoting anti-cancer immunity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Macrófagos/imunologia , Melanoma/imunologia , Monócitos/imunologia , Neoplasias Cutâneas/imunologia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Lectinas de Ligação a Manose/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Células Th2/imunologia
15.
APMIS ; 127(9): 642-652, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31274210

RESUMO

Hepatitis C virus (HCV) infection always leads to chronic hepatitis via dysregulation of host immunity. Notch signaling also modulates the response of monocytes/macrophages. Thus, we aimed to investigate the regulatory role of Notch signaling to CD14+ monocytes. Forty patients with chronic hepatitis C and twenty normal controls (NC) were enrolled. CD14+ monocytes and CD4+ T cells were purified from peripheral bloods. Notch receptors' mRNA expression in CD14+ monocytes was semi-quantified by real-time PCR. Cytokine production by CD14+ monocytes in response to γ-secretase inhibitor (GSI) was investigated by ELISA. GSI-induced CD14+ monocytes activity to HCV clearance in Huh7.5 cells and to CD4+ T cell differentiation was also assessed in direct and indirect contact co-culture system. Notch1 mRNA relative level was approximately 10-fold elevated in CD14+ monocytes from chronic hepatitis C patients when compared with NC. GSI stimulation resulted in enhanced cytokines production by CD14+ monocytes from chronic hepatitis C patients. GSI-stimulated CD14+ monocytes from chronic hepatitis C patients induced suppression of HCV RNA replication in both direct and indirect contact co-culture system of CD14+ monocytes and HCVcc-infected Huh7.5 cells, and this process was accompanied by elevation of interferon-γ production but not increased target cell death. Moreover, GSI stimulation also enhanced CD14+ monocytes-induced Th1 and Th17 cells activation, and this process required direct cell-to-cell contact. Effective antiviral therapy down-regulated Notch1 mRNA expression and promoted cytokine production by CD14+ monocytes from chronic hepatitis C. Current data revealed an important immunoregulatory property of Notch signaling to CD14+ monocytes in chronic HCV infection.


Assuntos
Hepatite C Crônica/imunologia , Monócitos/imunologia , Receptor Notch1/imunologia , Transdução de Sinais/imunologia , Adulto , Linfócitos T CD4-Positivos , Diferenciação Celular/imunologia , Técnicas de Cocultura , Regulação para Baixo/imunologia , Feminino , Hepacivirus/imunologia , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Células Th1/imunologia , Células Th17/imunologia , Adulto Jovem
16.
Nature ; 571(7764): 205-210, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270459

RESUMO

The mammalian brain contains neurogenic niches that comprise neural stem cells and other cell types. Neurogenic niches become less functional with age, but how they change during ageing remains unclear. Here we perform single-cell RNA sequencing of young and old neurogenic niches in mice. The analysis of 14,685 single-cell transcriptomes reveals a decrease in activated neural stem cells, changes in endothelial cells and microglia, and an infiltration of T cells in old neurogenic niches. T cells in old brains are clonally expanded and are generally distinct from those in old blood, which suggests that they may experience specific antigens. T cells in old brains also express interferon-γ, and the subset of neural stem cells that has a high interferon response shows decreased proliferation in vivo. We find that T cells can inhibit the proliferation of neural stem cells in co-cultures and in vivo, in part by secreting interferon-γ. Our study reveals an interaction between T cells and neural stem cells in old brains, opening potential avenues through which to counteract age-related decline in brain function.


Assuntos
Envelhecimento/fisiologia , Encéfalo/citologia , Movimento Celular , Células-Tronco Neurais/citologia , Neurogênese , Análise de Célula Única , Nicho de Células-Tronco/fisiologia , Linfócitos T/citologia , Animais , Sangue , Proliferação de Células , Células Clonais/citologia , Técnicas de Cocultura , Células Endoteliais/citologia , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Análise de Sequência de RNA , Transdução de Sinais , Linfócitos T/metabolismo , Transcriptoma/genética
17.
Bioresour Technol ; 290: 121796, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31319215

RESUMO

The aim of this study was to investigate the effects of steam explosion on lignocellulose digestibility of, and methane production from corn stover by a co-culture of anaerobic fungus and methanogen. The cumulative methane production at 72 h of incubation from the steam-exploded corn stover was 32.2 ±â€¯1.74 mL, which not significantly different (P > 0.05) from that of the untreated corn stover (37.1 ±â€¯1.09 mL). However, steam explosion decreased the hemicellulose contents of corn stover by 28.0 ±â€¯0.39% and increased the neutral detergent solute by 23.5 ±â€¯0.25%. While this treatment did not affect the dry matter digestibility (64.1 ±â€¯0.26%, and 64.1 ±â€¯0.28%, respectively). In conclusion, the co-culture of anaerobic fungus and methanogen can degrade the crude fibrous portion of corn stover without any pretreatments. It possesses promising biotechnological prospects for conversion of crop residue based straw resources to obtain biofuel in the form of methane.


Assuntos
Vapor , Zea mays , Anaerobiose , Técnicas de Cocultura , Explosões , Fungos , Lignina , Metano
18.
Zhonghua Gan Zang Bing Za Zhi ; 27(6): 450-456, 2019 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-31357762

RESUMO

Objective: To observe the effect of differentiated mature adipocytes on hepatic steatosis and aquaporin-9 (AQP9) expressions in HepG2 cells and further explore its possible mechanism of action. Methods: Human preadipocytes were cultured and differentiated to full maturity. HepG2 cells were co-cultured with non-differentiated adipocytes and differentiated mature adipocytes for 48 h, and then labeled as control group and experimental group. Oil red O staining and intracellular triglyceride content were performed on co-cultured HepG2 cells and simultaneous changes in phosphatidylinositol 3-kinase (PI3K) - serine/threonine kinase (Akt) signaling pathway, and AQP9 mRNA and protein levels were detected. The experimental group was co-cultured with recombinant human insulin-like growth factor-I (IGF-I), with the addition of 100ng/ml PI3K-Akt pathway agonist, labeled as experimental group + IGF-I group. The activation of PI3K-Akt pathway was verified by Western blotting (WB). The expression of AQP9 was detected by RT-q PCR and WB. The recombinant lentivirus LV-AQP9 or empty-loaded virus LV-PWPI was transfected with HepG2 cells by recombinant lentiviral transfection tecnique, and labeled as HepG2-AQP9 and HepG2-PWPI. The transfection efficiency was assessed by confocal laser scanning microscopy and RT-qPCR and WB detected the change of AQP9 expression level after virus transfection. Afterwards, the stable over-expressed HepG2-AQP9 cells and the empty-loaded HepG2-PWPI cells were co-cultured with differentiated mature adipocytes for 48h, and labeled as HepG2-AQP9 co-culture group, and then intracellular triglyceride content were detected with Oil red O staining. Finally, IGF-I was added to the HepG2-AQP9 co-culture group, which was recorded as HepG2-AQP9 co-culture + IGF-I group. Intracellular triglyceride content was detected with Oil red O staining, and WB verified PI3K-Akt signaling pathway activation and changes in AQP9 mRNA and protein levels. A t-test was used to compare the two independent samples. Results: The intracellular lipid droplets and triglyceride content (0.052 ± 0.005) in the experimental group was increased significantly than the control group (0.033 ± 0.003) (t= 5.225,P= 0.006), suggesting that adipocyte co-culture had induced steatosis in HepG2 cells. RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (3.615 ± 0.330) and protein levels (0.072 ± 0.005) in the experimental group were significantly higher than the control group (t= 13.708, 11.225,P= 0.005, < 0.001). WB results showed that the expression level of phosphorylated Akt (p-Akt) protein (0.116±0.003) in the experimental group was significantly lower than the control group (0.202 ± 0.003) (t= 27.136,P< 0.001). The total Akt protein was constant, and the p-Akt/total Akt (0.182 ± 0.017)was significantly lower than the control group (0.327 ± 0.019) (t= 2.431,P= 0.001), suggesting that adipocyte co-culture had inhibited PI3K- Akt signaling pathway in HepG2 cells and up-regulated the expression level of AQP9. WB results indicated that the expression level of p-Akt protein (0.194 ± 0.021) in the experimental group + IGF-I group was significantly higher than the experimental group (0.132 ± 0.003) (t= 5.082,P= 0.007). The total Akt protein was constant, and the p-Akt/total Akt (0.281 ± 0.009) was significantly higher than the control group (0.184 ± 0.132) (t= 10.311,P< 0.001). Simultaneously, RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (0.327 ± 0.347) and protein levels (0.042 ± 0.004) in the experimental group + IGF-I group were significantly lower than the experimental group (t= 33.573, 5.598,P< 0.001, 0.005), suggesting that adipocyte co-culture had possibility to regulate the expression level of AQP9 through the PI3K-Akt pathway. Confocal laser microscopy analysis showed that the transfection efficiency was more than 90%. RT-q PCR and WB results indicated that the expression levels of AQP9 mRNA and protein levels (0.373 ± 0.221) in HepG2-AQP9 group were significantly higher than HepG2-PWPI group (t=14.953, 28.931,P= 0.002 and 0.000), suggesting that the stable overexpression of AQP9 cell line was successfully constructed. The intracellular lipid droplets and triglyceride content in HepG2-AQP9 co-culture group was significantly increased (t= 5.478, 5.369,P= 0.005) than HepG2-PWPI co-culture group and HepG2-AQP9 co-culture+ IGF-I group, suggesting that the increased expression of AQP9 had promoted HepG2 steatosis in co-cultured adipocytes. WB results showed the expression levels of p-Akt protein (0.168 ± 0.006) and p-Akt/total Akt (0.265±0.009) in HepG2-AQP9 co-culture + IGF-1 group was significantly increased (t= 16.311, 8.769,P< 0.001) than HepG2-AQP9 co-culture group, while the expression levels of AQP9 mRNA (0.327 ± 0.034) and protein (0.375 ± 0.025) was significantly decreased (t= 33.573, 9.146,P< 0.001 and 0.001). Conclusion: Adipocytes co-culture can induce steatosis in HepG2 cells, and may participate in inhibiting PI3K-Akt signaling pathway to upregulate the expression of AQP9 in steatotic HepG2 cells.


Assuntos
Adipócitos , Aquaporinas , Regulação da Expressão Gênica no Desenvolvimento , Adipócitos/citologia , Adipócitos/metabolismo , Aquaporinas/genética , Técnicas de Cocultura , Células Hep G2 , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
19.
Life Sci ; 232: 116620, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31291594

RESUMO

AIMS: Cell-based biological pacemakers aim to overcome limitations and side effects of electronic pacemaker devices. We here developed and tested different approaches to achieve nodal-type differentiation using human adipose- and bone marrow-derived mesenchymal stem cells (haMSC, hbMSC). MAIN METHODS: haMSC and hbMSC were differentiated using customized protocols. Quantitative RT-PCR was applied for transcriptional pacemaker-gene profiling. Protein membrane expression was analyzed by immunocytochemistry. Pacemaker current (If) was studied in haMSC with and without lentiviral HCN4-transduction using patch clamp recordings. Functional characteristics were evaluated by co-culturing with neonatal rat ventricular myocytes (NRVM). KEY FINDINGS: Culture media-based differentiation for two weeks generated cells with abundant transcription of ion channel genes (Cav1.2, NCX1), transcription factors (TBX3, TBX18, SHOX2) and connexins (Cx31.9 and Cx45) characteristic for cardiac pacemaker tissue, but lack adequate HCN transcription. haMSC-derived cells revealed transcript levels, which were closer related to sinoatrial nodal cells than hbMSC-derived cells. To substitute for the lack of If, we performed lentiviral HCN4-transduction of haMSC resulting in stable If. Co-culturing with NRVM demonstrated that differentiated haMSC expressing HCN4 showed earlier onset of spontaneous contractions and higher beating regularity, synchrony and rate compared to co-cultures with non-HCN4-transduced haMSC or HCN4-transduced, non-differentiated haMSC. Confocal imaging indicated increased membrane expression of cardiac gap junctional proteins in differentiated haMSC. SIGNIFICANCE: By differentiation haMSC, rather than hbMSC attain properties favorable for cardiac pacemaking. In combination with lentiviral HCN4-transduction, a cellular phenotype was generated that sustainably controls and stabilizes rate in co-culture with NRVM.


Assuntos
Relógios Biológicos/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas Musculares/metabolismo , Canais de Potássio/metabolismo , Tecido Adiposo/fisiologia , Animais , Células da Medula Óssea/fisiologia , Diferenciação Celular/fisiologia , Técnicas de Cocultura , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células Musculares/metabolismo , Proteínas Musculares/fisiologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Ratos , Nó Sinoatrial
20.
Zhonghua Gan Zang Bing Za Zhi ; 27(7): 516-520, 2019 Jul 20.
Artigo em Chinês | MEDLINE | ID: mdl-31357777

RESUMO

Objective: To investigate the effects of different expression of monoacylglycerol lipase (MAGL) in tumor-associated macrophages (TAMs) with the proliferation of MHCC97H human liver cancer cells in vivo and its mechanism. Methods: Human peripheral blood-derived monocyte was induced to differentiate into M2-type TAMs and was identified by flow cytometry. The co-culture model of TAMs and MHCC97H human liver cancer cells was established, and the expression of MAGL in TAMs cells was detected by qRT-PCR. The expression of MAGL in TAMs cells was detected by plasmid transfection. ELISA and qRT-PCR was used to detect the mRNA expression levels and secretion levels of inflammatory factors in TAMs cells. The subcutaneous tumor model of MHCC97H mice was constructed to observe the effect of different expression of MAGL in TAMs cells with the proliferation of MHCC97H human liver cancer cells in vivo. F-test was used for the measurement of homogeneity of variance between two independent samples. A t-test was used for homogeneity of variance, and the corrected t-test was used for non-homogeneity of variance. Results: Human peripheral blood-derived monocytes were successfully induced to differentiate into M2-type TAMs. An in vitro co-culture model was established. qRT-PCR showed that MHCC97H human liver cancer cells significantly down-regulated the expressional level of MAGL in TAMs cells. The constructed subcutaneous tumor model of mice demonstrated that up-regulation up-regulation of MAGL expression in M2-type TAMs inhibited the proliferation of MHCC97H human liver cancer cells in vivo. Furthermore, the mechanistic study illustrated that the high expression of MAGL promoted the transcription and secretion of inflammatory factors such as interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha in M2-type TAMs cells. Conclusion: The overexpression of MAGL inhibits the proliferation of MHCC97H hepatocellular carcinoma cells in vivo, and its mechanism may be associated to the release of inflammatory factors that from TAMs cells.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Macrófagos/enzimologia , Monoacilglicerol Lipases/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Citocinas/metabolismo , Humanos , Camundongos
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