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1.
Exp Parasitol ; 210: 107842, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31978393

RESUMO

Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperature-dependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.


Assuntos
Acanthamoeba/fisiologia , Plaquetas/parasitologia , Meio Ambiente , Eritrócitos/parasitologia , Acanthamoeba/classificação , Acanthamoeba/genética , Acanthamoeba/patogenicidade , Difosfato de Adenosina/metabolismo , Doenças Transmissíveis Emergentes/parasitologia , Meios de Cultivo Condicionados , Eritrócitos/fisiologia , Genótipo , Hemólise , Humanos , Fagocitose , Agregação Plaquetária , Temperatura Ambiente , Trofozoítos/classificação , Trofozoítos/genética , Trofozoítos/patogenicidade , Trofozoítos/fisiologia
2.
Scand J Immunol ; 91(2): e12841, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31833575

RESUMO

Tumour-associated macrophages (TAMs) comprise an important part of the tumour microenvironment and play a key role in malignant tumours progression. Tumour-derived extracellular vesicles (EVs) modulated TAMs polarization and reprogrammed TAMs to influence the progression of various tumours. Here, we hypothesized that diffuse large B cell lymphoma (DLBCL)-derived EVs can regulate macrophages polarization and thus contribute to tumour progression. Our results demonstrated that EVs, released from DLBCL, augment the M2 polarization effects of macrophages. Moreover, conditional medium derived from macrophages by DLBCL-derived EVs stimulation revealed the superior effects of promoting tumour proliferation, migration and invasion. Further analysis demonstrated that DLBCL-derived EVs regulated the metabolism of macrophages by increasing the expression of PGC-1ß protein, thereby reprogramming the macrophage phenotype of promoting tumour progression. In conclusion, our findings signify that the DLBCL-derived EVs mediated the increasing of functional PGC-1ß protein in macrophages to promote the tumour progression.


Assuntos
Vesículas Extracelulares/metabolismo , Linfoma Difuso de Grandes Células B/imunologia , Macrófagos/fisiologia , Proteínas de Ligação a RNA/metabolismo , Carcinogênese , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Reprogramação Celular , Meios de Cultivo Condicionados , Progressão da Doença , Humanos , Ativação de Macrófagos , MicroRNAs/genética , Fenótipo , Microambiente Tumoral , Regulação para Cima
3.
Gut ; 69(2): 243-251, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31085554

RESUMO

OBJECTIVE: Cancer-associated fibroblasts (CAFs), a major component of cancer stroma, can confer aggressive properties to cancer cells by secreting multiple factors. Their phenotypes are stably maintained, but the mechanisms are not fully understood. We aimed to show the critical role of epigenetic changes in CAFs in maintaining their tumour-promoting capacity and to show the validity of the epigenomic approach in identifying therapeutic targets from CAFs to starve cancer cells. DESIGN: Twelve pairs of primary gastric CAFs and their corresponding non-CAFs (NCAFs) were established from surgical specimens. Genome-wide DNA methylation and H3K27me3 analyses were conducted by BeadArray 450K and ChIP-on-Chip, respectively. Functions of potential a therapeutic target were analysed by inhibiting it, and prognostic impact was assessed in a database. RESULTS: CAFs had diverse and distinct DNA methylation and H3K27me3 patterns compared with NCAFs. Loss of H3K27me3, but not DNA methylation, in CAFs was enriched for genes involved in stem cell niche, cell growth, tissue development and stromal-epithelial interactions, such as WNT5A, GREM1, NOG and IGF2. Among these, we revealed that WNT5A, which had been considered to be derived from cancer cells, was highly expressed in cancer stromal fibroblasts, and was associated with poor prognosis. Inhibition of secreted WNT5A from CAFs suppressed cancer cell growth and migration. CONCLUSIONS: H3K27me3 plays a crucial role in defining tumour-promoting capacities of CAFs, and multiple stem cell niche factors were secreted from CAFs due to loss of H3K27me3. The validity of the epigenetic approach to uncover therapeutic targets for cancer-starving therapy was demonstrated.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Neoplasias Gástricas/genética , Meios de Cultivo Condicionados , Metilação de DNA , DNA de Neoplasias/genética , Epigenômica/métodos , Ontologia Genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Histona Desmetilases com o Domínio Jumonji/deficiência , Mutação , Nicho de Células-Tronco , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas
4.
Biochemistry (Mosc) ; 84(11): 1375-1389, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760924

RESUMO

Mesenchymal stromal cell (MSCs) represent a class of biologics with the prospects for employment as immunomodulatory, tissue-protective, and regenerative therapeutics. In parallel with cellular therapy, cell-free therapy based on MSC-secreted bioactive factors is being actively developed. MSCs secrete a variety of protein, peptide, RNA, and lipid mediators which can be concentrated, frozen, or even lyophilized without loss of activity, which gives them a certain advantage over cellular products requiring liquid nitrogen storage and infrastructure to revive frozen cells. This review (i) describes currently conducted clinical trials of cell-free products containing MSC secretome; (ii) summarizes main approaches to the generation and characterization of conditioned media concentrates and extracellular vesicle isolates; (iii) analyzes a variety of preclinical studies where effectiveness of secretome products has been shown; and (iv) summarizes current knowledge about secretome bioactive components obtained by analysis of in vivo models testing the therapeutic potential of the MSC secretome.


Assuntos
Meios de Cultivo Condicionados/química , Células-Tronco Mesenquimais/metabolismo , Lesão Renal Aguda/patologia , Lesão Renal Aguda/prevenção & controle , Animais , Artrite/patologia , Artrite/prevenção & controle , Células da Medula Óssea/citologia , Meios de Cultivo Condicionados/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exossomos/metabolismo , Lesão Pulmonar/patologia , Lesão Pulmonar/prevenção & controle , Células-Tronco Mesenquimais/citologia
5.
J Environ Pathol Toxicol Oncol ; 38(2): 173-183, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31679280

RESUMO

In the present study, we investigated the effects of conditioned media (CM) collected from the cancer cell lines (K562, MCF-7, and HeLa) on peripheral blood mononuclear cells (PBMCs) isolated from the healthy human blood. The soluble factors in the CM are probably responsible for the differential mRNA expressions of Foxp3, Helios, Neuropilin- 1 (NRP-1), and glycoprotein A repetitions predominant (GARP), along with IFN-γ and TGF-ß in PBMCs cultured with cancer cells CM. The PBMCs cultured with CM of K562 showed increased expression of Foxp3, Helios, NRP-1, GARP, IFN-γ, and TGF-ß compared to PBMCs cultured with CM of MCF-7 and HeLa cells. In addition, the intracellular staining on PBMCs cultured with CM from cell lines were also evaluated for CD4, CD25, Foxp3, Helios, and NRP-1 by multicolor flow cytometry. The expression of CD4+CD25+Foxp3+, CD4+Helios+Foxp3+ and CD+NRP-1+Foxp3+ showed retarded cell population compared to control PBMCs. Our data suggest that soluble factors in CM of cancer cells may trigger the immune response in PBMCs resulting in a systematic response. Further research could lead to the identification of specific soluble factors that are involved in trafficking of cells into the immune cascades, which could be a safe and promising strategy for targeting human cancers.


Assuntos
Expressão Gênica , Interferon gama/genética , Leucócitos Mononucleares/metabolismo , Fator de Crescimento Transformador beta/genética , Meios de Cultivo Condicionados , Células HeLa , Humanos , Interferon gama/metabolismo , Células K562 , Células MCF-7 , Fator de Crescimento Transformador beta/metabolismo
6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(9): 769-775, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31750816

RESUMO

Objective To explore the functions and mechanisms of macrophages derived from PGRN gene knockout (PGRN-/- ) C57BL/6 mice in the invasion and migration of breast cancer cells. Methods Breast cancer cells were cultured in conditioned medium of macrophages derived from WT and PGRN-/- mice. TranswellTM assay and scratch assay were used to detect the invasion and migration ability of cancer cells. Western blot analysis was used to detect the expression of E-cadherin and N-cadherin in cancer cells. Cytokine array, real-time quantitative PCR and ELISA were performed to investigate the differences of cytokines secreted by macrophages derived from WT and PGRN-/- mice. Breast cancer cells were treated by the differentially expressed cytokine interleukin-6 (IL-6), and then the above methods were used to investigate its effect on cancer cells. Western blot analysis was used to verify the roles of NF-κB and JAK/STAT3 signaling pathways. Results The macrophages derived from PGRN-/- mice blocked NF-κB signaling pathway, reduced IL-6 secretion, and inhibited the invasion and migration of breast cancer cells. IL-6 activated JAK/STAT3 signaling pathway to promote the invasion and migration of breast cancer cells. Conclusion The macrophages derived from PGRN-/- mice can block the NF-κB and JAK/STAT3 signaling pathways, down-regulate IL-6 expression, and inhibit the invasion and migration of breast cancer cells.


Assuntos
Neoplasias da Mama/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interleucina-6/imunologia , Macrófagos/imunologia , Transdução de Sinais , Animais , Neoplasias da Mama/imunologia , Caderinas , Movimento Celular , Meios de Cultivo Condicionados , Granulinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Invasividade Neoplásica , Fator de Transcrição STAT3/metabolismo , Células Tumorais Cultivadas
7.
Exp Eye Res ; 189: 107860, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31655040

RESUMO

Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPARγ2. ADSCs could differentiate into neural cells, stained positive to ß-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.


Assuntos
Adipócitos/citologia , Doenças da Córnea/terapia , Ceratócitos da Córnea/citologia , Criopreservação , Células-Tronco/citologia , Adulto , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Doenças da Córnea/patologia , Meios de Cultivo Condicionados/farmacologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Transplante de Células-Tronco/métodos
8.
J Immunol Res ; 2019: 3616120, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31565660

RESUMO

Immune cell therapy has emerged as a promising approach to treat malignancies that were up until recently only treated on a palliative basis. Chimeric antigen receptor- (CAR-) modified T lymphocytes (T cells) in particular have proven to be very effective for certain hematological malignancies. The production of CAR T cells usually involves viral transduction and ex vivo culture of T cells. The aim of this study was to explore the use of human platelet lysate (HPL) compared to two commonly used supplements, human AB serum (ABS) and fetal bovine serum (FBS), for modified T cell production. For studying transduction, activated T cells were transduced with lentivirus to deliver GFP transgenes with three different promoters. Transduction efficiency (percent GFP) was similar among the supplements, and a modest increase in the transgene product (mean fluorescence intensity) was observed when HPL was used as a supplement compared to ABS. To study the effect of supplements on expansion, peripheral blood mononuclear cells (PBMCs) were activated and expanded in the presence of interleukin 2 (IL2) for fourteen days. T cell expansions using HPL and ABS were comparable and slightly less than the expansion obtained with FBS. Interestingly, cells expanded in media supplemented with HPL showed a higher percentage of T cells with a central memory phenotype compared to those expanded in ABS or FBS. Protein profiling revealed that the phenotypic differences may be explained by elevated levels of several cytokines in HPL, including IL7. The results suggest that the use of HPL as a cell culture supplement during the production of modified T cells is a reasonable alternative to ABS. Furthermore, the use of HPL may enhance in vivo performance of the final product by enriching for central memory T cells that are associated with long-term persistence following adoptive transfer.


Assuntos
Plaquetas/metabolismo , Meios de Cultivo Condicionados/farmacologia , Memória Imunológica/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Técnicas de Cultura de Células , Biologia Computacional/métodos , Citocinas/metabolismo , Expressão Gênica , Ontologia Genética , Genes Reporter , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Proteoma , Proteômica , Linfócitos T/metabolismo , Transdução Genética , Transgenes
9.
Int J Mol Sci ; 20(19)2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31569434

RESUMO

Injecting human mesenchymal stem cells (hMSCs) at wound sites is known to have a therapeutic effect; however, hMSCs have several limitations, such as low viability and poor engraftment after injection, as well as a potential risk of oncogenesis. The use of a conditioned medium (CM) was suggested as an alternative method for treating various wounds instead of direct hMSC administration. In addition to not having the adverse effects associated with hMSCs, a CM can be easily mass produced and can be stored for long-term, thereby making it useful for clinical applications. In general, a CM is collected from hMSCs with low passage number; whereas, the hMSCs with high passage number are usually discarded because of their low therapeutic efficacy as a result of reduced angiogenic factor secretion. Herein, we used a CM collected from high passage number (passage 12, P12) hMSCs treated with gold-iron nanoparticles (AuFe NPs). Our AuFe NPs were designed to release the iron ion intracellularly via endocytosis. Endosomes with low pH can dissolve iron from AuFe NPs, and thus, the intracellularly released iron ions up-regulate the hypoxia-inducible factor 1α and vascular endothelial growth factor (VEGF) expression. Through this mechanism, AuFe NPs improve the amount of VEGF expression from P12 hMSCs so that it is comparable to the amount of VEGF expression from low passage number (passage 6, P6), without treatment. Furthermore, we injected the CM retrieved from P12 MSCs treated with AuFe NPs in the mouse skin wound model (AuFe P12 group). AuFe P12 group revealed significantly enhanced angiogenesis in the mouse skin wound model compared to the high passage hMSC CM-injected group. Moreover, the result from the AuFe P12 group was similar to that of the low passage hMSC CM-injected group. Both the AuFe P12 group and low passage hMSC CM-injected group presented significantly enhanced re-epithelization, angiogenesis, and tissue remodeling compared to the high passage hMSC CM-injected group. This study reveals a new strategy for tissue regeneration based on CM injection without considering the high cell passage count.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Células-Tronco Mesenquimais/metabolismo , Nanopartículas , Cicatrização/efeitos dos fármacos , Materiais Biocompatíveis/química , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Nanopartículas/química , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Tissue Cell ; 60: 48-59, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31582018

RESUMO

Malignant Melanoma is known for being one of the most aggressive cancers with an incidence that increases every year. Cancer stem cells (CSCs) are involved in the resistance to therapeutic treatments, the metastatic process, and the patient's relapses. Thus, it is of vital importance for researchers to find the methodology that allows us to obtain enriched subpopulations that maintain their stem-like properties without differentiating over time. In the present manuscript, our objective was to compare the ability of conditioned medium obtained from human mesenchymal stem cells (MSCs) isolated by enzymatic and non-enzymatic methods for the enrichment and maintenance of melanoma CSCs. Our results showed for the first time that MSCs isolated by less aggressive methodology displayed higher CSCs enrichment and maintenance capacity. Because they do not undergo enzymatic and proteolytic stress, MSCs produce a greater amount of signalling molecules, helping to improve the phenotype characteristics of CSCs and keep them over time.


Assuntos
Meios de Cultivo Condicionados/química , Melanoma/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Cutâneas/patologia , Linhagem Celular Tumoral , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Cultura Primária de Células
11.
Int J Nanomedicine ; 14: 7475-7488, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571859

RESUMO

Background: Wear particle-induced inflammatory osteolysis and the consequent aseptic loosening constitute the leading reasons for prosthesis failure and revision surgery. Several studies have demonstrated that the macrophage polarization state and immune response play critical roles in periprosthetic osteolysis and tissue repair, but the immunomodulatory role of lithium chloride (LiCl), which has a protective effect on wear particle-induced osteolysis by suppressing osteoclasts and attenuating inflammatory responses, has never been investigated. Methods: In this work, the immunomodulatory capability of LiCl on titanium (Ti) nanoparticle-stimulated transformation of macrophage phenotypes and the subsequent effect on osteogenic differentiation were investigated. We first speculated that LiCl attenuated Ti nanoparticle-stimulated inflammation responses by driving macrophage polarization and generating an immune micro-environment to improve osteogenesis. Furthermore, a metal nanoparticle-stimulated murine air pouch inflammatory model was applied to confirm this protective effect in vivo. Results: The results revealed that metal nanoparticles significantly activate M1 phenotype (proinflammatory macrophage) expression and increase proinflammatory cytokines secretions in vitro and in vivo, whereas LiCl drives macrophages to the M2 phenotype (anti-inflammatory macrophage) and increases the release of anti-inflammatory and bone-related cytokines. This improved the osteogenic differentiation capability of rat bone marrow mesenchymal stem cells (rBMSCs). In addition, we also provided evidence that LiCl inhibits the phosphorylation of the p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways in wear particle-treated macrophages. Conclusion: LiCl has the immunomodulatory effects to alleviate Ti nanoparticle-mediated inflammatory reactions and enhance the osteogenic differentiation of rBMSCs by driving macrophage polarization. Thus, LiCl may be an effective therapeutic alternative for preventing and treating wear debris-induced inflammatory osteolysis.


Assuntos
Fatores Imunológicos/farmacologia , Inflamação/patologia , Cloreto de Lítio/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nanopartículas Metálicas/química , Osteogênese/efeitos dos fármacos , Titânio/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Ratos
12.
Nat Commun ; 10(1): 4481, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578371

RESUMO

Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Luminescentes/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Trimetoprima/farmacologia , Animais , Anti-Infecciosos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Microscopia Confocal
13.
J Microbiol Biotechnol ; 29(11): 1830-1840, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31601058

RESUMO

Loliolide is one of the most ubiquitous monoterpenoid compounds found in algae, and its potential therapeutic effect on various dermatological conditions via agent-induced biological functions, including anti-oxidative and anti-apoptotic properties, was demonstrated. Here, we investigated the effects of loliolide on hair growth in dermal papilla (DP) cells, the main components regulating hair growth and loss conditions. For this purpose, we used a threedimensional (3D) DP spheroid model that mimics the in vivo hair follicle system. Biochemical assays showed that low doses of loliolide increased the viability and size of 3D DP spheroids in a dose-dependent manner. This result correlated with increases in expression levels of hair growth-related autocrine factors including VEGF, IGF-1, and KGF. Immunoblotting and luciferase-reporter assays further revealed that loliolide induced AKT phosphorylation, and this effect led to stabilization of ß-catenin, which plays a crucial role in the hair-inductive properties of DP cells. Further experiments showed that loliolide increased the expression levels of the DP signature genes, ALP, BMP2, VCAN, and HEY1. Furthermore, conditioned media from loliolide-treated DP spheroids significantly enhanced proliferation and the expression of hair growth regulatory genes in keratinocytes. These results suggested that loliolide could function in the hair growth inductivity of DP cells via the AKT/ ß-catenin signaling pathways.


Assuntos
Benzofuranos/farmacologia , Derme/efeitos dos fármacos , Folículo Piloso/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Derme/citologia , Células HEK293 , Folículo Piloso/crescimento & desenvolvimento , Humanos , Queratinócitos/efeitos dos fármacos , Monoterpenos/farmacologia , Fosforilação/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos
14.
Nature ; 574(7779): 553-558, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645721

RESUMO

Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5-8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain 'activated fibroblasts' that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals.


Assuntos
Envelhecimento/metabolismo , Reprogramação Celular , Senescência Celular/fisiologia , Fibroblastos/metabolismo , Cicatrização , Animais , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mediadores da Inflamação/metabolismo , Judeus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Processos Estocásticos , Fatores de Tempo , Cicatrização/efeitos dos fármacos
15.
Breast Cancer Res ; 21(1): 109, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31533776

RESUMO

BACKGROUND: Bone morphogenetic proteins (BMPs) have been reported to maintain epithelial integrity and to antagonize the transforming growth factor ß (TGFß)-induced epithelial to mesenchymal transition. The expression of soluble BMP antagonists is dysregulated in cancers and interrupts proper BMP signaling in breast cancer. METHODS: In this study, we mined the prognostic role of BMP antagonists GREMLIN 1 (GREM1) in primary breast cancer tissues using in-house and publicly available datasets. We determined which cells express GREM1 RNA using in situ hybridization (ISH) on a breast cancer tissue microarray. The effects of Grem1 on the properties of breast cancer cells were assessed by measuring the mesenchymal/stem cell marker expression and functional cell-based assays for stemness and invasion. The role of Grem1 in breast cancer-associated fibroblast (CAF) activation was measured by analyzing the expression of fibroblast markers, phalloidin staining, and collagen contraction assays. The role of Grem1 in CAF-induced breast cancer cell intravasation and extravasation was studied by utilizing xenograft zebrafish breast cancer (co-) injection models. RESULTS: Expression analysis of clinical breast cancer datasets revealed that high expression of GREM1 in breast cancer stroma is correlated with a poor prognosis regardless of the molecular subtype. The large majority of human breast cancer cell lines did not express GREM1 in vitro, but breast CAFs did express GREM1 both in vitro and in vivo. Transforming growth factor ß (TGFß) secreted by breast cancer cells, and also inflammatory cytokines, stimulated GREM1 expression in CAFs. Grem1 abrogated bone morphogenetic protein (BMP)/SMAD signaling in breast cancer cells and promoted their mesenchymal phenotype, stemness, and invasion. Moreover, Grem1 production by CAFs strongly promoted the fibrogenic activation of CAFs and promoted breast cancer cell intravasation and extravasation in co-injection xenograft zebrafish models. CONCLUSIONS: Our results demonstrated that Grem1 is a pivotal factor in the reciprocal interplay between breast cancer cells and CAFs, which promotes cancer cell invasion. Targeting Grem1 could be beneficial in the treatment of breast cancer patients with high Grem1 expression.


Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Mamárias Experimentais , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Fosforilação , Prognóstico , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra
16.
World J Gastroenterol ; 25(33): 4892-4903, 2019 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-31543681

RESUMO

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapy is currently under study to treat inflammatory bowel diseases. MSC bioactive products could represent a valid alternative to overcome issues associated with systemic whole-cell therapies. However, MSC anti-inflammatory mechanisms differ between rodents and humans, impairing the reliability of preclinical models. AIM: To evaluate the effect of conditioned medium (CM) derived from porcine vascular wall MSCs (pVW-MSCs) on survival and differentiation of porcine and guinea pig enteric ganglia exposed to lipopolysaccharide (LPS). METHODS: Primary cultures of enteric ganglia were obtained by mechanic and enzymatic digestion of ileum resections from guinea pigs (Cavia porcellus) (GPEG) and pigs (Suus scrofa) (PEG). pVW-MSCs were derived by enzymatic digestion from vascular wall resections of porcine aorta and tested by immunoflowcytometry for MSC immune profile. Enteric ganglia were treated with increasing concentrations of LPS, CM derived by pVW-MSCs or a combination of CM and LPS 1 µg/mL. Cell count and morphometric analysis of HuD positive neurons and glial fibrillary acidic protein positive glial cells were performed by immunofluorecent staining of cultured ganglia. RESULTS: PEG showed a higher number of neurons compared to GPEG. Overall, CM exerted a protective role on LPS-treated enteric ganglia. CM in combination with LPS increased the number of glial cells per ganglion in both cultures evoking glial cells differentiation in porcine cultures. CONCLUSION: These findings suggest an immunomodulating activity of pVW-MSCs mediators on the enteric nervous system in inflammatory conditions.


Assuntos
Sistema Nervoso Entérico/efeitos dos fármacos , Vesículas Extracelulares/imunologia , Células-Tronco Mesenquimais/metabolismo , Neuroglia/efeitos dos fármacos , Animais , Vasos Sanguíneos/citologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/imunologia , Vesículas Extracelulares/metabolismo , Cobaias , Humanos , Íleo/irrigação sanguínea , Íleo/imunologia , Íleo/inervação , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/terapia , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/imunologia , Mucosa Intestinal/inervação , Lipopolissacarídeos/toxicidade , Masculino , Transplante de Células-Tronco Mesenquimais , Neuroglia/imunologia , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Cultura Primária de Células , Sus scrofa
17.
Int J Mol Sci ; 20(18)2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31547264

RESUMO

The transplantation of Wharton's jelly derived mesenchymal stromal cells (WJ-MSCs) possesses therapeutic potential for the treatment of a spinal cord injury (SCI). Generally, the main effect of MSCs is mediated by their paracrine potential. Therefore, application of WJ-MSC derived conditioned media (CM) is an acknowledged approach for how to bypass the limited survival of transplanted cells. In this study, we compared the effect of human WJ-MSCs and their CM in the treatment of SCI in rats. WJ-MSCs and their CM were intrathecally transplanted in the three consecutive weeks following the induction of a balloon compression lesion. Behavioral analyses were carried out up to 9 weeks after the SCI and revealed significant improvement after the treatment with WJ-MSCs and CM, compared to the saline control. Both WJ-MSCs and CM treatment resulted in a higher amount of spared gray and white matter and enhanced expression of genes related to axonal growth. However, only the CM treatment further improved axonal sprouting and reduced the number of reactive astrocytes in the lesion area. On the other hand, WJ-MSCs enhanced the expression of inflammatory and chemotactic markers in plasma, which indicates a systemic immunological response to xenogeneic cell transplantation. Our results confirmed that WJ-MSC derived CM offer an alternative to direct stem cell transplantation for the treatment of SCI.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Traumatismos da Medula Espinal/terapia , Geleia de Wharton/citologia , Animais , Células Cultivadas , Citocinas/sangue , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Ratos , Ratos Wistar , Traumatismos da Medula Espinal/sangue , Traumatismos da Medula Espinal/fisiopatologia
18.
Life Sci ; 235: 116817, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476309

RESUMO

AIMS: In the tumor microenvironment, dysregulated immune cells could promote tumor progression, invasion and metastasis, by establishing a symbiotic relationship with cancer cells. A pivotal role is played by monocyte recruitment and induction of tumor-associated macrophages (TAMs), which provide immunosuppression and tumorigenesis. The effect of nemorosone, an antiproliferative phytocomponent present in Cuban Propolis, on TAM-induced tumor progression remains to be elucidated. Here we investigated the symbiotic relationship between monocytic leukemia THP-1 and hepatocellular carcinoma HepG2 cells, and the role of nemorosone in preventing TAM-induced tumor growth. MAIN METHODS: Macrophage differentiation induced by HepG2-conditioned medium was assessed by flow cytometry, analysis of secreted molecules and cytokine expression. The effect of nemorosone and/or conditioned THP-1-medium on HepG2 proliferation was evaluated by MTT assay, colony formation, cells cycle and migration assays. KEY FINDINGS: HepG2 cells induced THP-1 recruitment and differentiation to macrophages. When compared with control THP-1 cells, differentiated THP-1 showed a significant increase of the matrix metalloproteinases MMP-2 and MMP-9 expression (P < 0.01), and slightly induced HepG2 cells growth. This effect was counteracted by nemorosone, which also significantly inhibited colony formation (P < 0.01) and migratory capacity of HepG2 cells, driving a high percentage of cells (80%) to the G0/G1 phase. SIGNIFICANCE: HepG2-conditioned medium is a suitable model for THP-1 modulation and differentiation. Moreover, nemorosone significantly inhibits the proliferation of HepG2 cells, both in presence and absence of the soluble factors secreted by TAMs. Further studies are needed to elucidate the role of this natural compound in the HCC-TAM relationship.


Assuntos
Benzofenonas/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Monócitos/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Diferenciação Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Monócitos/citologia , Monócitos/metabolismo , Células THP-1
19.
Int J Oncol ; 55(4): 949-959, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485602

RESUMO

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite advances in surgery, radiotherapy and chemotherapy, the overall 5­year survival rate of patients with OSCC has not significantly improved. In addition, the prognosis of patients with advanced­stage OSCC remains poor. Therefore, it is necessary to develop novel therapeutic modalities. Vincristine (VCR), a naturally occurring vinca alkaloid, is a classical microtubule­destabilizing agent and is widely used in the treatment of a number of cancers. Despite the proven antitumor benefits of VCR treatment, one of the major reasons for the failure of treatment is drug resistance. Changes in the tumor microenvironment are responsible for cross­talk between cells, which may facilitate drug resistance in cancers; secreted proteins may promote communication between cancer cells to induce the development of resistance. To identify the secreted proteins involved in VCR resistance, conditioned media was obtained, and an antibody array was conducted to screen a comprehensive secretion profile between VCR­resistant (SAS­VCR) and parental (SAS) OSCC cell lines. The results showed that amphiregulin (AREG) was highly expressed and secreted in SAS­VCR cells. Pretreatment with exogenous recombinant AREG markedly increased drug resistance against VCR in OSCC cells, as assessed by an MTT assay. Colony formation, MTT and western blot assays were performed to investigate the effects of AREG knockdown on VCR sensitivity. The results indicated that AREG expression can regulate VCR resistance in OSCC cells; overexpression of AREG increased VCR resistance in parental cells, whereas AREG knockdown decreased the VCR resistance of resistant cells. In addition, it was also demonstrated that the glycogen synthase kinase­3ß pathway may be involved in AREG­induced VCR resistance. These findings may provide rationale to combine VCR with blockade of AREG­related pathways for the effective treatment of OSCC.


Assuntos
Anfirregulina/metabolismo , Carcinoma de Células Escamosas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Bucais/metabolismo , Anfirregulina/genética , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Prognóstico , Transdução de Sinais , Regulação para Cima , Vincristina/farmacologia
20.
Int J Med Sci ; 16(8): 1157-1170, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31523179

RESUMO

Background: Current opinion suggests that expansion of cancer stem cells (CSCs) and activation of pro-tumoral inflammation cascade correlate with cancer progression. Materials and methods: We explored the possible contributions of MRC-5 cancer-associated fibroblasts to the expression profiles of CSC markers and inflammation-associated cell surface molecules. The liver cancer cell lines Bel-7402, SMMC-7721, MHCC-LM3, and HepG2 cultured in conditioned medium (CM) from MRC-5 served as test groups, whereas the liver cancer cell lines cultured in normal medium served as control groups. Results: Flow cytometry revealed that the proportions of CD90+ cells were significantly higher in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, and moderately higher in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, than in controls. The CD90+/CD45- proportions were elevated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, but reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, as compared to controls. Western blotting indicated that Nanog was downregulated in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls; that POU5F1 (OCT4/3) was downregulated in MHCC-LM3-(MRC-5)-CM, but upregulated in Bel-7402-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls, and that CK19 was upregulated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, compared to controls. Proportions of cells expressing Toll-like receptor-1+ (TLR1) and TLR4 were significantly higher in MHCC-LM3-(MRC-5)-CM cells, and moderately higher in HepG2-(MRC-5)-CM cells, than controls. However, the TLR1+ and TLR4+ proportions were lower in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells than controls. Proportions of CD25+ cells were reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, but elevated in MHCC-LM3-(MRC-5)-CM and Bel-7402-(MRC-5)-CM cells, compared to controls. Proportion of CD61+ cells was higher in liver cancer cells cultured in MRC-5-CM than in controls. Proportion of CD14+ cells was lower in HCC cells cultured in MRC-5-CM than in controls. Conclusion: MRC-5 extensively affected the production of CSC markers and inflammation-associated cell surface molecules. Tumor-targeting molecular therapies should consider these findings.


Assuntos
Biomarcadores Tumorais/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Inflamação/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Citometria de Fluxo , Células Hep G2 , Humanos , Integrina beta3/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 1 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Microambiente Tumoral , Ensaio Tumoral de Célula-Tronco
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