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1.
Anticancer Res ; 41(9): 4229-4238, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475042

RESUMO

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) shows poor survival and early systemic dissemination. Cancer associated fibroblasts (CAFs) enhance migration and invasion of cancer cells. We aimed to investigate the role of CAFs in cell migration and their underlying paracrine effects. MATERIALS AND METHODS: Using Transwell® migration assays, PDAC cells (PANC-1) and three distinct types of fibroblasts were analyzed: CAFs, genetically transformed human foreskin-fibroblasts (BJeLR), and non-transformed human foreskin-fibroblasts (VH7). IL6 in the culture supernatant was measured to investigate paracrine communication in monocultures and direct/indirect cocultures. RESULTS: CAFs showed a significantly higher capacity to migrate in vitro when compared to benign fibroblasts (p=0.009). They also facilitated the migration of PDAC cells in coculture (p=0.001). Neither BJeLR, nor VH7 displayed such features. This was accompanied by a significant increase in IL-6 when CAFs were cocultured with PANC-1 (p=0.009). CONCLUSION: CAFs are a key element of intra-tumoral migration and should be further investigated as a potential therapeutic target.


Assuntos
Fibroblastos Associados a Câncer/citologia , Carcinoma Ductal Pancreático/patologia , Prepúcio do Pênis/citologia , Interleucina-6/metabolismo , Neoplasias Pancreáticas/patologia , Fibroblastos Associados a Câncer/imunologia , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/imunologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Feminino , Prepúcio do Pênis/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/imunologia , Comunicação Parácrina , Microambiente Tumoral
2.
Anticancer Res ; 41(9): 4249-4258, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34475044

RESUMO

BACKGROUND/AIM: Recent studies have indicated the clinical significance of tumor-associated macrophages (TAMs) in breast cancer; however, the detailed mechanisms of cell-cell interactions between TAMs and cancer cells remain unclear. MATERIALS AND METHODS: In vitro cell culture studies using human monocyte-derived macrophages and breast cancer cell lines were performed to test which cytokines would be involved in cell-cell interactions between cancer cells and macrophages. In addition, studies using human resected samples and animal breast cancer models were performed to examine the significance of TAMs in cancer development. RESULTS: Osteopontin, HB-EGF, and IL-6 were suggested to be macrophage-derived growth factors for breast cancer cells. FROUNT inhibitor significantly blocked TAM infiltration and subcutaneous tumor growth in an E0771 mouse breast cancer model. CONCLUSION: TAMs express growth factors, such as osteopontin, for cancer cells, and targeting of TAM infiltration might be a promising approach for anti-breast cancer therapy.


Assuntos
Neoplasias da Mama/patologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Interleucina-6/genética , Macrófagos/citologia , Osteopontina/genética , Macrófagos Associados a Tumor/citologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Interleucina-6/metabolismo , Células MCF-7 , Macrófagos/metabolismo , Camundongos , Transplante de Neoplasias , Osteopontina/metabolismo , Microambiente Tumoral , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia
3.
Food Res Int ; 147: 110545, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34399522

RESUMO

Understanding the role of food-related factors on the efficacy of protective cultures is essential to attain optimal results for developing biopreservation-based strategies. The aim of this work was to assess and model growth of Latilactobacillus sakei CTC494 and Listeria monocytogenes CTC1034, and their interaction, in two different ready-to-eat fish products (i.e., surimi-based product and tuna pâté) at 2 and 12 °C. The existing expanded Jameson-effect and a new expanded Jameson-effect model proposed in this study were evaluated to quantitatively describe the effect of microbial interaction. The inhibiting effect of the selected lactic acid bacteria strain on the pathogen growth was product dependent. In surimi product, a reduction of lag time of both strains was observed when growing in coculture at 2 °C, followed by the inhibition of the pathogen when the bioprotective L. sakei CTC494 reached the maximum population density, suggesting a mutualism-antagonism continuum phenomenon between populations. In tuna pâté, L. sakei CTC494 exerted a strong inhibition of L. monocytogenes at 2 °C (<0.5 log increase) and limited the growth at 12 °C (<2 log increase). The goodness-of-fit indexes indicated that the new expanded Jameson-effect model performed better and appropriately described the different competition patterns observed in the tested fish products. The proposed expanded competition model allowed for description of not only antagonistic but also mutualism-based interactions based on their influence on lag time.


Assuntos
Lactobacillales , Listeria monocytogenes , Animais , Técnicas de Cocultura , Produtos Pesqueiros , Interações Microbianas
4.
Nutrients ; 13(8)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34444982

RESUMO

Several natural compounds, such as vitamin K2, have been highlighted for their positive effects on bone metabolism. It has been proposed that skeletal disorders, such as osteoporosis, may benefit from vitamin K2-based therapies or its regular intake. However, further studies are needed to better clarify the effects of vitamin K2 in bone disorders. To this aim, we developed in vitro a three-dimensional (3D) cell culture system one step closer to the bone microenvironment based on co-culturing osteoblasts and osteoclasts precursors obtained from bone specimens and peripheral blood of the same osteoporotic patient, respectively. Such a 3-D co-culture system was more informative than the traditional 2-D cell cultures when responsiveness to vitamin K2 was analyzed, paving the way for data interpretation on single patients. Following this approach, the anabolic effects of vitamin K2 on the osteoblast counterpart were found to be correlated with bone turnover markers measured in osteoporotic patients' sera. Overall, our data suggest that co-cultured osteoblasts and osteoclast precursors from the same osteoporotic patient may be suitable to generate an in vitro 3-D experimental model that potentially reflects the individual's bone metabolism and may be useful to predict personal responsiveness to nutraceutical or drug molecules designed to positively affect bone health.


Assuntos
Osso e Ossos/efeitos dos fármacos , Nutrientes/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoporose , Medicina de Precisão/métodos , Vitamina K 2/farmacologia , Biomarcadores/sangue , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/metabolismo , Células Cultivadas , Técnicas de Cocultura/métodos , Feminino , Humanos , Masculino , Modelos Biológicos , Nutrientes/uso terapêutico , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Modelagem Computacional Específica para o Paciente , Vitamina K 2/uso terapêutico , Vitaminas/farmacologia , Vitaminas/uso terapêutico
5.
Nat Biomed Eng ; 5(8): 847-863, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34385693

RESUMO

The therapeutic efficacy of stem cells transplanted into an ischaemic brain depends primarily on the responses of the neurovascular unit. Here, we report the development and applicability of a functional neurovascular unit on a microfluidic chip as a microphysiological model of ischaemic stroke that recapitulates the function of the blood-brain barrier as well as interactions between therapeutic stem cells and host cells (human brain microvascular endothelial cells, pericytes, astrocytes, microglia and neurons). We used the model to track the infiltration of a number of candidate stem cells and to characterize the expression levels of genes associated with post-stroke pathologies. We observed that each type of stem cell showed unique neurorestorative effects, primarily by supporting endogenous recovery rather than through direct cell replacement, and that the recovery of synaptic activities is correlated with the recovery of the structural and functional integrity of the neurovascular unit rather than with the regeneration of neurons.


Assuntos
AVC Isquêmico/terapia , Dispositivos Lab-On-A-Chip , Transplante de Células-Tronco , Astrócitos/citologia , Astrócitos/metabolismo , Barreira Hematoencefálica/química , Barreira Hematoencefálica/metabolismo , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Microglia/citologia , Microglia/metabolismo , Microvasos/citologia , Modelos Biológicos , Neurônios/citologia , Neurônios/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
6.
Talanta ; 234: 122702, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364499

RESUMO

In this work, an integrated 3-dimensional microfluidic device was developed for simulation of the immune microenvironment of glioma niche through the co-culture of three kinds of related cells. Glioma cells, endothelial cells and macrophages were co-cultured together in the microfluidic device, spatially separated by the design of a coffer structure and the use of hydrogel. This platform enabled separate monitoring of the morphology change and migration of cells, as well as molecular interactions between different kinds of cells. Tumor cells were found to exhibit EMT like shape change to become thinner, and sensitive perception and taxis toward macrophages. The influence of tumor cells and the microenvironment, macrophages would be re-educated and the phenotype could be changed from M1 (tumor-suppressive) to M2 (tumor-supportive), which could be validated through cytokines analysis. This 3D microfluidic tumor model provides a powerful tool for studying the biological properties of glioma niche.


Assuntos
Glioma , Microfluídica , Biomimética , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Endoteliais , Humanos , Fenótipo , Microambiente Tumoral
7.
Int J Mol Sci ; 22(15)2021 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-34360675

RESUMO

In recent decades, interest in natural compounds has increased exponentially due to their numerous beneficial properties in the treatment of various acute and chronic diseases. A group of plant derivatives with great scientific interest is terpenic compounds. Among the plants richest in terpenes, the genus Ferula L. is one of the most representative, and ferutinin, the most common sesquiterpene, is extracted from the leaves, rhizome, and roots of this plant. As reported in the scientific literature, ferutinin possesses antioxidant and anti-inflammatory properties, as well as valuable estrogenic properties. Neurodegenerative and demyelinating diseases are devastating conditions for which a definite cure has not yet been established. The mechanisms involved in these diseases are still poorly understood, and oxidative stress is considered to be both a key modulator and a common denominator. In the proposed experimental system, co-cultured human neurons (SH-SY5Y) and human oligodendrocytes (MO3.13) were treated with the pro-inflammatory agent lipopolysaccharide at a concentration of 1 µg/mL for 24 h or pretreated with ferutinin (33 nM) for 24 h and subsequently exposed to lipopolysaccharide 1 µg/mL for 24 h. Further studies would, however, be needed to establish whether this natural compound can be used as a support strategy in pathologies characterized by progressive inflammation and oxidative stress phenomena.


Assuntos
Benzoatos/farmacologia , Cicloeptanos/farmacologia , Inflamação/tratamento farmacológico , Lipopolissacarídeos/toxicidade , Neurônios/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Estresse Oxidativo , Sesquiterpenos/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Linhagem Celular , Técnicas de Cocultura , Escherichia coli , Humanos , Inflamação/induzido quimicamente , Neurônios/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Substâncias Protetoras/farmacologia
8.
Nat Commun ; 12(1): 4997, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404774

RESUMO

Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro.


Assuntos
Agregação Celular/fisiologia , Técnicas de Cocultura , Miócitos Cardíacos/fisiologia , Família Aldeído Desidrogenase 1/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteína Morfogenética Óssea 4 , Cálcio/metabolismo , Diferenciação Celular , Genes do Tumor de Wilms , Humanos , Células-Tronco Pluripotentes Induzidas , Fator de Crescimento Insulin-Like II/metabolismo , Mesoderma , Miócitos de Músculo Liso , Retinal Desidrogenase/metabolismo , Semaforinas , Células-Tronco , Proteínas com Domínio T/metabolismo
9.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360750

RESUMO

Tissue regeneration depends on the complex processes of angiogenesis, inflammation and wound healing. Regarding muscle tissue, glucocorticoids (GCs) inhibit pro-inflammatory signalling and angiogenesis and lead to muscle atrophy. Our hypothesis is that the synthetic GC dexamethasone (dex) impairs angiogenesis leading to muscle atrophy or inhibited muscle regeneration. Therefore, this study aims to elucidate the effect of dexamethasone on HUVECs under different conditions in mono- and co-culture with myoblasts to evaluate growth behavior and dex impact with regard to muscle atrophy and muscle regeneration. Viability assays, qPCR, immunofluorescence as well as ELISAs were performed on HUVECs, and human primary myoblasts seeded under different culture conditions. Our results show that dex had a higher impact on the tube formation when HUVECs were maintained with VEGF. Gene expression was not influenced by dex and was independent of cells growing in a 2D or 3D matrix. In co-culture CD31 expression was suppressed after incubation with dex and gene expression analysis revealed that dex enhanced expression of myogenic transcription factors, but repressed angiogenic factors. Moreover, dex inhibited the VEGF mediated pro angiogenic effect of myoblasts and inhibited expression of angiogenic inducers in the co-culture model. This is the first study describing a co-culture of human primary myoblast and HUVECs maintained under different conditions. Our results indicate that dex affects angiogenesis via inhibition of VEGF release at least in myoblasts, which could be responsible not only for the development of muscle atrophy after dex administration, but also for inhibition of muscle regeneration after vascular damage.


Assuntos
Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mioblastos Esqueléticos/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Mioblastos Esqueléticos/citologia
10.
Theriogenology ; 173: 211-220, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34399385

RESUMO

Although it is known that stresses on females damage oocytes with increased production of stress hormones, whether corticotrophin-releasing hormone (CRH) or adrenocorticotropic hormone (ACTH) harm oocytes directly are largely unknown. We demonstrated that CRH exposure during in vitro maturation impaired competence of both pig and mouse cumulus-oocyte-complexes (COCs), and it impaired competence and induced apoptosis in pig cumulus-denuded oocytes (DOs) but not in mouse DOs. CRH receptor 1 was expressed in pig DOs and in cumulus cells (CCs) of both species but not in mouse DOs. In the presence of CRH, whereas mouse CCs underwent apoptosis, pig CCs did not. While pig CCs did, mouse CCs did not express CRH-binding protein. ACTH did not affect competence of either pig or mouse COCs or DOs although they all expressed ACTH receptor. Both pig and mouse CCs expressed steroidogenic acute regulatory protein (StAR), and ACTH enhanced their progesterone production while alleviating their apoptosis. Neither pig nor mouse DOs expressed StAR, but ACTH inhibited maturation-promoting factor and decelerated meiotic progression of DOs suggesting activation of protein kinase A (PKA). In conclusion, CRH impaired pig and mouse oocyte competence by interacting with CRH receptor and inducing CCs apoptosis, respectively. ACTH activated PKA in both DOs and CCs although it showed no effect on oocyte competence.


Assuntos
Hormônio Adrenocorticotrópico , Hormônio Liberador da Corticotropina , Hormônio Adrenocorticotrópico/farmacologia , Animais , Técnicas de Cocultura/veterinária , Células do Cúmulo , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Camundongos , Oócitos , Suínos
11.
Stem Cell Res Ther ; 12(1): 440, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362454

RESUMO

BACKGROUND: Autologous adipose tissue transfer may be performed for aesthetic needs following the resection of dermatofibrosarcoma protuberans (DFSP), the most common cutaneous soft tissue sarcoma, excluding Kaposi sarcoma. The regenerative effectiveness of cell-assisted lipotransfer is dependent on the presence of adipose tissue-derived stem cells (ADSCs). This is the first study to evaluate the potential oncological risks as ADSCs could unintentionally be sited within the proximity of the tumor microenvironment of DFSP cells. METHODS: Primary DFSP cells were indirectly co-cultured with ADSCs in a conditioned medium or in a Transwell system. The impact was analyzed by assessing proliferation, migration, invasion, angiogenesis, and tumor-associated genes and proteins. Results of these assays were compared between co-culture and mono-culture conditions. RESULTS: Our experimental results showed that ADSCs were able to promote proliferation, migration, invasion, and angiogenesis of DFSP cells; this was accompanied by a significant increase in the expression levels of beta-type platelet-derived growth factor receptor, collagen type I alpha 1 chain, vascular endothelial growth factor, hepatocyte growth factor, and basic fibroblast growth factor. CONCLUSIONS: The current report clearly demonstrates that ADSCs can enhance different malignant properties of DFSP cells in vitro, which should not be neglected when considering the clinical use of human ADSCs and its related derivatives in skin regenerative therapies.


Assuntos
Dermatofibrossarcoma , Neoplasias Cutâneas , Tecido Adiposo , Proliferação de Células , Técnicas de Cocultura , Dermatofibrossarcoma/genética , Dermatofibrossarcoma/terapia , Humanos , Neoplasias Cutâneas/terapia , Células-Tronco , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular
12.
Front Immunol ; 12: 692729, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34421902

RESUMO

Epidemiological studies and clinical trials suggest Bacillus Calmette-Guérin (BCG) vaccine has protective effects against coronavirus disease 2019 (COVID-19). There are now over 30 clinical trials evaluating if BCG vaccination can prevent or reduce the severity of COVID-19. However, the mechanism by which BCG vaccination can induce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses is unknown. Here, we identify 8 novel BCG-derived peptides with significant sequence homology to either SARS-CoV-2 NSP3 or NSP13-derived peptides. Using an in vitro co-culture system, we show that human CD4+ and CD8+ T cells primed with a BCG-derived peptide developed enhanced reactivity to its corresponding homologous SARS-CoV-2-derived peptide. As expected, HLA differences between individuals meant that not all persons developed immunogenic responses to all 8 BCG-derived peptides. Nevertheless, all of the 20 individuals that were primed with BCG-derived peptides developed enhanced T cell reactivity to at least 7 of 8 SARS-CoV-2-derived peptides. These findings provide an in vitro mechanism that may account, in part, for the epidemiologic observation that BCG vaccination confers some protection from COVID-19.


Assuntos
Vacina BCG/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas , SARS-CoV-2/imunologia , Adulto , COVID-19/imunologia , COVID-19/prevenção & controle , Células Cultivadas , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Humanos , Masculino , Análise de Sequência de Proteína , Homologia de Sequência , Vacinas de Subunidades/imunologia , Adulto Jovem
13.
Nat Commun ; 12(1): 5029, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413303

RESUMO

Dendritic cells (DC) in the lung that induce Th17 differentiation remain incompletely understood, in part because conventional CD11b+ DCs (cDC2) are heterogeneous. Here, we report a population of cDCs that rapidly accumulates in lungs of mice following house dust extract inhalation. These cells are Ly-6C+, are developmentally and phenotypically similar to cDC2, and strongly promote Th17 differentiation ex vivo. Single cell RNA-sequencing (scRNA-Seq) of lung cDC2 indicates 5 distinct clusters. Pseudotime analysis of scRNA-Seq data and adoptive transfer experiments with purified cDC2 subpopulations suggest stepwise developmental progression of immature Ly-6C+Ly-6A/E+ cDC2 to mature Ly-6C-CD301b+ lung resident cDC2 lacking Ccr7 expression, which then further mature into CD200+ migratory cDC2 expressing Ccr7. Partially mature Ly-6C+Ly-6A/E-CD301b- cDC2, which express Il1b, promote Th17 differentiation. By contrast, CD200+ mature cDC2 strongly induce Th2, but not Th17, differentiation. Thus, Th17 and Th2 differentiation are promoted by lung cDC2 at distinct stages of maturation.


Assuntos
Asma/imunologia , Antígeno CD11b/imunologia , Células Dendríticas/imunologia , Pulmão/imunologia , Células Th17/imunologia , Células Th2/imunologia , Transferência Adotiva/métodos , Animais , Asma/metabolismo , Asma/patologia , Sequência de Bases , Antígeno CD11b/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Célula Única/métodos , Células Th17/citologia , Células Th2/citologia
14.
Environ Sci Technol ; 55(14): 10142-10151, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34196176

RESUMO

Direct interspecies electron transfer (DIET) between microbial species prevails in some key microbial consortia. However, the electron transfer mechanism(s) in these consortia is controversial due to lack of efficient characterization methods. Here, we provide an in situ anaerobic spectroelectrochemical coculture cell (in situ ASCC) to induce the formation of DIET coculture biofilm on the interdigitated microelectrode arrays and characterize the electron transfer directly. Two typical Geobacter DIET cocultures, Geobacter metallireducens and wild-type Geobacter sulfurreducens (G.m&G.s) and G. metallireducens and a G. sulfurreducens strain deficient in citrate synthase (G.m&G.s-ΔgltA), were selected. In situ Raman and electrochemical Fourier transform infrared (FTIR) spectroscopy indicated that cytochromes are abundant in the electric syntrophic coculture. Cyclic voltammetry and potential step experiment revealed a diffusion-controlled electron transfer process and the electrochemical gating measurements further demonstrated a cytochrome-mediated electron transfer in the DIET coculture. Furthermore, the G.m&G.s-ΔgltA coculture displayed a higher redox conductivity than the G.m&G.s coculture, consistent with the existence of an intimate and efficient electrical connection between these two species. Our findings provide the first report of a redox-gradient-driven electron transport facilitated by c-type cytochromes in DIET coculture, supporting the model that DIET is mediated by cytochromes and suggest a platform to explore the other DIET consortia.


Assuntos
Geobacter , Técnicas de Cocultura , Citocromos/metabolismo , Transporte de Elétrons , Geobacter/metabolismo , Oxirredução
15.
Int J Mol Sci ; 22(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299050

RESUMO

The role of astrocytes in the periphery of metastatic brain tumors is unclear. Since astrocytes regulate central nervous metabolism, we hypothesized that changes in astrocytes induced by contact with cancer cells would appear in the metabolome of both cells and contribute to malignant transformation. Coculture of astrocytes with breast cancer cell supernatants altered glutamate (Glu)-centered arginine-proline metabolism. Similarly, the metabolome of cancer cells was also altered by astrocyte culture supernatants, and the changes were further amplified in astrocytes exposed to Glu. Inhibition of Glu uptake in astrocytes reduces the variability in cancer cells. Principal component analysis of the cancer cells revealed that all these changes were in the first principal component (PC1) axis, where the responsible metabolites were involved in the metabolism of the arginine-proline, pyrimidine, and pentose phosphate pathways. The contribution of these changes to the tumor microenvironment needs to be further pursued.


Assuntos
Astrócitos/patologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Metaboloma , Microambiente Tumoral/imunologia , Animais , Animais Recém-Nascidos , Apoptose , Astrócitos/imunologia , Astrócitos/metabolismo , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Camundongos , Ratos
16.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299012

RESUMO

Atopic dermatitis (AD) represents a severe global burden on physical, physiological and mental health. Innate immune cell basophils are essential for provoking allergic inflammation in AD. However, the roles of novel immunoregulatory cytokine IL-37 in basophils remain elusive. We employed in vitro co-culture of human basophils and human keratinocyte HaCaT cells and an in vivo MC903-induced AD murine model to investigate the anti-inflammatory mechanism of IL-37. In the in vitro model, IL-37b significantly decreased Der p1-induced thymic stromal lymphopoietin (TSLP) overexpression in HaCaT cells and decreased the expression of TSLP receptor as well as basophil activation marker CD203c on basophils. IL-37 could also reduce Th2 cytokine IL-4 release from TSLP-primed basophils ex vivo. In the in vivo model, alternative depletion of basophils ameliorated AD symptoms and significantly lowered the Th2 cell and eosinophil populations in the ear and spleen of the mice. Blocking TSLP alleviated the AD-like symptoms and reduced the infiltration of basophils in the spleen. In CRISPR/Cas9 human IL-37b knock-in mice or mice with direct treatment by human IL-37b antibody, AD symptoms including ear swelling and itching were significantly alleviated upon MC903 challenge. Notably, IL-37b presence significantly reduced the basophil infiltration in ear lesions. In summary, IL-37b could regulate the TSLP-mediated activation of basophils and reduce the release of IL-4. The results, therefore, suggest that IL-37 may target TSLP-primed basophils to alleviate AD.


Assuntos
Basófilos/imunologia , Citocinas/metabolismo , Dermatite Atópica/metabolismo , Interleucina-1/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Basófilos/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Citocinas/antagonistas & inibidores , Citocinas/farmacologia , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/genética , Dermatite Atópica/imunologia , Regulação para Baixo , Orelha/patologia , Eosinófilos/metabolismo , Técnicas de Introdução de Genes , Humanos , Interleucina-1/genética , Interleucina-1/farmacologia , Interleucina-1/uso terapêutico , Interleucina-4/metabolismo , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Baço/imunologia , Baço/metabolismo , Células Th2/imunologia , Regulação para Cima
17.
FASEB J ; 35(8): e21791, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34320240

RESUMO

Chemical neurotransmission typically occurs through synapses. Previous ultrastructural examinations of monoamine neuron axon terminals often failed to identify a pre- and postsynaptic coupling, leading to the concept of "volume" transmission. Whether this results from intrinsic properties of these neurons remains undefined. We find that dopaminergic neurons in vitro establish a distinctive axonal arbor compared to glutamatergic or GABAergic neurons in both size and propensity of terminals to avoid direct contact with target neurons. While most dopaminergic varicosities are active and contain exocytosis proteins like synaptotagmin 1, only ~20% of these are synaptic. The active zone protein bassoon was found to be enriched in dopaminergic terminals that are in proximity to a target cell. Finally, we found that the proteins neurexin-1αSS4- and neuroligin-1A+B play a critical role in the formation of synapses by dopamine (DA) neurons. Our findings suggest that DA neurons are endowed with a distinctive developmental connectivity program.


Assuntos
Axônios/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Corpo Estriado/citologia , Dopamina/metabolismo , Neurônios Dopaminérgicos/fisiologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Moléculas de Adesão Celular Neuronais/genética , Diferenciação Celular , Técnicas de Cocultura/métodos , Dopamina/genética , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
18.
Methods Mol Biol ; 2352: 133-148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324185

RESUMO

Astrocytes are essential cells for normal brain functionality and have recently emerged as key players in many neurological diseases. However, the limited availability of human primary astrocytes for cell culture studies hinders our understanding of their physiology and precise role in disease development and progression. Here, we describe a detailed step-by-step protocol to rapidly and efficiently generate functionally mature induced astrocytes (iAs) from human embryonic and induced pluripotent stem cells (hES/iPSCs). Astrocyte induction is accomplished by ectopic lentiviral expression of two gliogenic transcription factors, Sox9 and Nfib. iAs exhibit morphology features as well as gene and protein expression similar to human mature astrocytes and display important astrocytic functions, such as glutamate uptake, propagation of calcium waves, expression of various cytokines after stimulation, and support of synapse formation and function, making them suitable models for studying the role of astrocytes in health and disease. Moreover, we describe a procedure for cryopreservation of iAs for long-term storage or shipping. Finally, we provide the required information needed to set up cocultures with human induced neurons (iNs, also described in this book), generated from hES/iPSCs, to generate cocultures, allowing studies on astrocyte-neuron interactions and providing new insights in astrocyte-associated disease mechanisms.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Reprogramação Celular/genética , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Técnicas de Cultura de Células , Células Cultivadas , Técnicas de Reprogramação Celular , Técnicas de Cocultura , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica
19.
Methods Mol Biol ; 2352: 253-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324192

RESUMO

Adult human cortical organotypic slice culture is an attractive model system to explore mechanisms of human brain pathology as well as to test drug candidates for treatment of neurodegeneration. Acute studies in human brain slices are limited by the lifetime of the tissue and focus mainly on hippocampus slice preparation. Here we describe the derivation of human organotypic slice cultures of cortical origin, which can be kept in culture for up to 6 weeks. This method enabled us to test the system in coculture with reprogrammed neurons and show its feasibility in neuronal cell integration experiments in human-to-human grafting situation.


Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular , Córtex Cerebral/citologia , Técnicas de Cocultura , Neurônios/citologia , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Cultura de Células , Humanos
20.
Am J Physiol Lung Cell Mol Physiol ; 321(3): L518-L532, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34231378

RESUMO

Macrophages are critical regulators of pulmonary fibrosis. Their plasticity, proximity, and ability to cross talk with structural cells of the lung make them a key cell type of interest in the regulation of lung fibrosis. Macrophages can express a variety of phenotypes, which have been historically represented through an "M1-like" to "M2-like" delineation. In this classification, M1-like macrophages are proinflammatory and have increased phagocytic capacity compared with alternatively activated M2-like macrophages that are profibrotic and are associated with wound healing. Extensive evidence in the field in both patients and animal models aligns pulmonary fibrosis with M2 macrophages. In this study, we performed RNA sequencing (RNAseq) to fully characterize M1- vs. M2-skewed bone marrow-derived macrophages (BMDMs) and investigated the profibrotic abilities of M2 BMDM conditioned media (CM) to promote fibroblast migration and proliferation, alveolar epithelial cell (AEC) apoptosis, and mRNA expression of key fibrotic genes in both fibroblasts and AECs. Although M2 CM-treated fibroblasts had increased migration and M2 CM-treated fibroblasts and AECs had increased expression of profibrotic proteins over M1 CM-treated cells, all differences can be attributed to M2 polarization reagents IL-4 and IL-13 also present in the CM. Collectively, these data suggest that the profibrotic effects associated with M2 macrophage CM in vitro are attributable to effects of polarization cytokines rather than additional factors secreted in response to those polarizing cytokines.


Assuntos
Células Epiteliais Alveolares/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Macrófagos/metabolismo , Fibrose Pulmonar/metabolismo , RNA-Seq , Células Epiteliais Alveolares/patologia , Animais , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Feminino , Fibroblastos/patologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia
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