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1.
Nat Commun ; 12(1): 4730, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354063

RESUMO

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Dispositivos Lab-On-A-Chip , Neurogênese/fisiologia , Organoides/crescimento & desenvolvimento , Organoides/fisiologia , Animais , Encéfalo/citologia , Meios de Cultura , Fenômenos Eletrofisiológicos , Matriz Extracelular/fisiologia , Estudos de Viabilidade , Perfilação da Expressão Gênica , Humanos , Hidrogéis , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Anatômicos , Modelos Neurológicos , Neurogênese/genética , Neuroglia/citologia , Neuroglia/fisiologia , Técnicas de Cultura de Órgãos/instrumentação , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Suínos
2.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445380

RESUMO

Cholangiocarcinoma (CC) is an aggressive malignancy with an inferior prognosis due to limited systemic treatment options. As preclinical models such as CC cell lines are extremely rare, this manuscript reports a protocol of cholangiocarcinoma patient-derived organoid culture as well as a protocol for the transition of 3D organoid lines to 2D cell lines. Tissue samples of non-cancer bile duct and cholangiocarcinoma were obtained during surgical resection. Organoid lines were generated following a standardized protocol. 2D cell lines were generated from established organoid lines following a novel protocol. Subcutaneous and orthotopic patient-derived xenografts were generated from CC organoid lines, histologically examined, and treated using standard CC protocols. Therapeutic responses of organoids and 2D cell lines were examined using standard CC agents. Next-generation exome and RNA sequencing was performed on primary tumors and CC organoid lines. Patient-derived organoids closely recapitulated the original features of the primary tumors on multiple levels. Treatment experiments demonstrated that patient-derived organoids of cholangiocarcinoma and organoid-derived xenografts can be used for the evaluation of novel treatments and may therefore be used in personalized oncology approaches. In summary, this study establishes cholangiocarcinoma organoids and organoid-derived cell lines, thus expanding translational research resources of cholangiocarcinoma.


Assuntos
Antineoplásicos/administração & dosagem , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/genética , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Organoides/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Neoplasias dos Ductos Biliares/genética , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos/métodos , Organoides/efeitos dos fármacos , Organoides/patologia , Organoides/transplante , Medicina de Precisão , Análise de Sequência de RNA , Células Tumorais Cultivadas , Sequenciamento Completo do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
3.
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
4.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298973

RESUMO

Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.


Assuntos
Simulação por Computador , Mucosa Intestinal/crescimento & desenvolvimento , Intestino Delgado/crescimento & desenvolvimento , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptores Acoplados a Proteínas G/genética
5.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201564

RESUMO

Obesity increases the risk of hip osteoarthritis (OA). Recent studies have shown that adipokine extracellular nicotinamide phosphoribosyltransferase (eNAMPT or visfatin) induces the production of IL-6 and matrix metalloproteases (MMPs) in chondrocytes, suggesting it may promote articular cartilage degradation. However, neither the functional effects of extracellular visfatin on human articular cartilage tissue, nor its expression in the joint of hip OA patients of varying BMI, have been reported. Hip OA joint tissues were collected from patients undergoing joint replacement surgery. Cartilage explants were stimulated with recombinant human visfatin. Pro-inflammatory cytokines and MMPs were measured by ELISA and Luminex. Localisation of visfatin expression in cartilage tissue was determined by immunohistochemistry. Cartilage matrix degradation was determined by quantifying proteoglycan release. Expression of visfatin was elevated in the synovial tissue of hip OA patients who were obese, and was co-localised with MMP-13 in areas of cartilage damage. Visfatin promoted the degradation of hip OA cartilage proteoglycan and induced the production of pro-inflammatory cytokines (IL-6, MCP-1, CCL20, and CCL4) and MMPs. The elevated expression of visfatin in the obese hip OA joint, and its functional effects on hip cartilage tissue, suggests it plays a central role in the loss of cartilage integrity in obese patients with hip OA.


Assuntos
Cartilagem Articular/patologia , Citocinas/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Osteoartrite do Quadril/metabolismo , Idoso , Idoso de 80 Anos ou mais , Cartilagem Articular/metabolismo , Quimiocinas/metabolismo , Condrócitos/metabolismo , Citocinas/sangue , Articulação do Quadril/metabolismo , Articulação do Quadril/fisiopatologia , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/sangue , Obesidade/metabolismo , Técnicas de Cultura de Órgãos , Osteoartrite do Quadril/patologia , Proteoglicanas/metabolismo
6.
Int J Mol Sci ; 22(14)2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-34299287

RESUMO

Organoids represent one of the most important advancements in the field of stem cells during the past decade. They are three-dimensional in vitro culturing models that originate from self-organizing stem cells and can mimic the in vivo structural and functional specificities of body organs. Organoids have been established from multiple adult tissues as well as pluripotent stem cells and have recently become a powerful tool for studying development and diseases in vitro, drug screening, and host-microbe interaction. The use of stem cells-that have self-renewal capacity to proliferate and differentiate into specialized cell types-for organoids culturing represents a major advancement in biomedical research. Indeed, this new technology has a great potential to be used in a multitude of fields, including cancer research, hereditary and infectious diseases. Nevertheless, organoid culturing is still rife with many challenges, not limited to being costly and time consuming, having variable rates of efficiency in generation and maintenance, genetic stability, and clinical applications. In this review, we aim to provide a synopsis of pluripotent stem cell-derived organoids and their use for disease modeling and other clinical applications.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Animais , Humanos , Modelos Biológicos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo
7.
Methods Mol Biol ; 2277: 405-421, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080165

RESUMO

The more recent studies of human pathologies have essentially revealed the complexity of the interactions involved at the different levels of integration in organ physiology. Integrated organ thus reveals functional properties not predictable by underlying molecular events. It is therefore obvious that current fine molecular analyses of pathologies should be fruitfully combined with integrative approaches of whole organ function. It follows that an important issue in the comprehension of the link between molecular events in pathologies and whole organ function/dysfunction is the development of new experimental strategies aimed at the study of the integrated organ physiology. Cardiovascular diseases are a good example as heart submitted to ischemic conditions has to cope both with a decreased supply of nutrients and oxygen, and the necessary increased activity required to sustain whole body-including the heart itself-oxygenation.By combining the principles of control analysis with noninvasive 31P NMR measurement of the energetic intermediates and simultaneous measurement of heart contractile activity, we developed MoCA (for Modular Control and regulation Analysis), an integrative approach designed to study in situ control and regulation of cardiac energetics during contraction in intact beating perfused isolated heart (Diolez et al., Am J Physiol Regul Integr Comp Physiol 293(1):R13-R19, 2007). Because it gives real access to integrated organ function, MoCA brings out a new type of information-the "elasticities," referring to integrated internal responses to metabolic changes-that may be a key to the understanding of the processes involved in pathologies. MoCA can potentially be used not only to detect the origin of the defects associated with the pathology, but also to provide the quantitative description of the routes by which these defects-or also drugs-modulate global heart function, therefore opening therapeutic perspectives. This review presents selected examples of the applications to isolated intact beating heart that evidence different modes of energetic regulation of cardiac contraction. We also discuss the clinical application by using noninvasive 31P cardiac energetics examination under clinical conditions for detection of heart pathologies.


Assuntos
Metabolismo Energético , Espectroscopia de Ressonância Magnética/métodos , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Animais , Cálcio/metabolismo , Cardiotônicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Epinefrina/metabolismo , Cobaias , Coração/efeitos dos fármacos , Homeostase , Humanos , Masculino , Mitocôndrias Cardíacas/metabolismo , Miofibrilas/metabolismo , Técnicas de Cultura de Órgãos/métodos , Ratos , Simendana/farmacologia
8.
Phytomedicine ; 88: 153589, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111617

RESUMO

BACKGROUND: Irritable bowel syndrome (IBS) is a functional bowel disorder, in which recurrent abdominal pain is associated with defecation or a change in bowel habits. STW 5-II is a combination of six medicinal herbs with a clinically proven efficacy in managing IBS. AIM: This study aims to establish an in vitro IBS model using mouse intestinal organoids and to explore the anti-inflammatory and tight junction protective activities of the multi-herbal preparation STW 5-II. METHODS: Intestinal organoids were cultured in 1:1 Matrigel™ and medium domes. Inflammation and tight junction disruption were induced by a cocktail of cytokines (TNFα, IFNγ, IL-1ß, IL-6) and bacterial proteins (LPS, flagellin). Organoids were treated with different concentrations of STW 5-II, and its multi-target activity was assessed using microarray analyses, RT-qPCR, immunofluorescence, western blot, immunohistochemistry, and a FITC permeability assay. In addition, we analyzed the expression of pNF-κB, pSTAT1, iNOS and ZO-1. In silico analyses were conducted to predict and identify the active components that may be responsible in mediating the multi-target anti-inflammatory activity of STW 5-II. RESULTS: An organoid based IBS model was successfully established. STW 5-II effectively reduced the cytokines-induced overexpression of the pro-inflammatory mediators pNF-κB, pSTAT1 and iNOS. Moreover, STW 5-II attenuated cytokine-mediated downregulation of the tight junction protein, ZO-1. This finding was confirmed by a FITC permeability assay. In silico analyses revealed a promising inhibitory activity of some isolated compounds from STW 5-II against NF-κB, STAT1 and iNOS. CONCLUSION: STW 5-II possesses multiple anti-inflammatory as well as tight junction protective activities that could explain its clinically proven efficacy in managing IBS symptoms.


Assuntos
Anti-Inflamatórios/farmacologia , Intestinos/efeitos dos fármacos , Síndrome do Intestino Irritável/tratamento farmacológico , Extratos Vegetais/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Simulação por Computador , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Síndrome do Intestino Irritável/etiologia , Camundongos , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos , Organoides/metabolismo , Organoides/fisiopatologia , Extratos Vegetais/química , Fator de Transcrição STAT1/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
9.
Toxicol Lett ; 349: 124-133, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34153409

RESUMO

With a possibility for the use of chemical weapons in battlefield or in terrorist activities, effective therapies against the devastating ocular injuries, from their exposure, are needed. Oxygen plays a vital role in ocular tissue preservation and wound repair. We tested the efficacy of supersaturated oxygen emulsion (SSOE) in reducing ex vivo corneal and keratocyte injury from chloropicrin (CP). CP, currently used as a pesticide, is a chemical threat agent like the vesicating mustard agents and causes severe corneal injury. Since our previous study in human corneal epithelial cells showed the treatment potential of SSOE (55 %), we further tested its efficacy in an ex vivo CP-induced rabbit corneal injury model. Corneas were exposed to CP (700 nmol) for 2 h, washed and cultured with or without SSOE for 24 h or 96 h. At 96 h post CP exposure, SSOE treatment presented a healing tendency of the corneal epithelial layer, and abrogated the CP-induced epithelial apoptotic cell death. SSOE treatment also reduced the CP induced DNA damage (H2A.X phosphorylation) and inflammatory markers (e.g. MMP9, IL-21, MIP-1ß, TNFα). Further examination of the treatment efficacy of SSOE alone or in combination with other therapies in in vivo cornea injury models for CP and vesicants, is warranted.


Assuntos
Queimaduras Químicas/tratamento farmacológico , Córnea/efeitos dos fármacos , Queimaduras Oculares/tratamento farmacológico , Hidrocarbonetos Clorados/toxicidade , Oxigênio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Queimaduras Químicas/etiologia , Queimaduras Químicas/metabolismo , Queimaduras Químicas/patologia , Córnea/metabolismo , Córnea/patologia , Citocinas/metabolismo , Dano ao DNA , Emulsões , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/metabolismo , Queimaduras Oculares/patologia , Mediadores da Inflamação/metabolismo , Masculino , Técnicas de Cultura de Órgãos , Coelhos , Cicatrização/efeitos dos fármacos
10.
Nat Commun ; 12(1): 3372, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099734

RESUMO

Despite advances in the development of highly effective androgen receptor (AR)-directed therapies for the treatment of men with advanced prostate cancer, acquired resistance to such therapies frequently ensues. A significant subset of patients with resistant disease develop AR-negative tumors that lose their luminal identity and display neuroendocrine features (neuroendocrine prostate cancer (NEPC)). The cellular heterogeneity and the molecular evolution during the progression from AR-positive adenocarcinoma to AR-negative NEPC has yet to be characterized. Utilizing a new genetically engineered mouse model, we have characterized the synergy between Rb1 loss and MYCN (encodes N-Myc) overexpression which results in the formation of AR-negative, poorly differentiated tumors with high metastatic potential. Single-cell-based approaches revealed striking temporal changes to the transcriptome and chromatin accessibility which have identified the emergence of distinct cell populations, marked by differential expression of Ascl1 and Pou2f3, during the transition to NEPC. Moreover, global DNA methylation and the N-Myc cistrome are redirected following Rb1 loss. Altogether, our data provide insight into the progression of prostate adenocarcinoma to NEPC.


Assuntos
Adenocarcinoma/genética , Carcinoma Neuroendócrino/genética , Regulação Neoplásica da Expressão Gênica , Próstata/metabolismo , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Adenocarcinoma/metabolismo , Animais , Carcinoma Neuroendócrino/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Técnicas de Cultura de Órgãos/métodos , Prognóstico , Próstata/patologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo
11.
Expert Opin Drug Metab Toxicol ; 17(8): 937-946, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33945374

RESUMO

Introduction: Recent studies suggested that extracellular vesicles (EVs) play a role both in the metastatic niche formation and in the progression of several tumors, including pancreatic cancer. In particular, the effects of EVs on metastasis should be studied in model systems that take into account both the tumor cells and the metastatic site/tumor microenvironment. Studies with labeled EVs or EV-secreting cells in ex vivo models will reflect the physiological and pathological functions of EVs. The organotypic-tissue slide culture systems can fulfill such a role.Areas covered: This review provides an overview of available organotypic-culture slide systems. We specifically focus on the assay system of liver culture-slides in combination with pancreatic tumors, which can be modulated to test the efficacy of new therapeutic approaches.Expert opinion: The intercellular exchange of EVs has emerged as a biologically relevant phenomenon to drive cancer metastasis. However, further models need to be developed to better elucidate the functional roles of EVs. The use of novel organotypic slide culture systems provides the opportunity to explore the role of EVs in the metastatic behavior of pancreatic cancer, decreasing the use of costly and cumbersome organoid or animal models.


Assuntos
Modelos Biológicos , Técnicas de Cultura de Órgãos , Neoplasias Pancreáticas/patologia , Animais , Vesículas Extracelulares/metabolismo , Humanos , Fígado/citologia , Metástase Neoplásica , Neoplasias Pancreáticas/terapia
12.
Nat Commun ; 12(1): 2992, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016976

RESUMO

Rapid death of infected cells is an important antiviral strategy. However, fast decisions that are based on limited evidence can be erroneous and cause unnecessary cell death and subsequent tissue damage. How cells optimize their death decision making strategy to maximize both speed and accuracy is unclear. Here, we show that exposure to TNF, which is secreted by macrophages during viral infection, causes cells to change their decision strategy from "slow and accurate" to "fast and error-prone". Mathematical modeling combined with experiments in cell culture and whole organ culture show that the regulation of the cell death decision strategy is critical to prevent HSV-1 spread. These findings demonstrate that immune regulation of cellular cognitive processes dynamically changes a tissues' tolerance for self-damage, which is required to protect against viral spread.


Assuntos
Apoptose/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Córnea/imunologia , Córnea/virologia , Modelos Animais de Doenças , Feminino , Herpes Simples/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Microscopia Intravital , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Modelos Imunológicos , Células NIH 3T3 , Técnicas de Cultura de Órgãos , Cultura Primária de Células , Imagem com Lapso de Tempo , Fator de Necrose Tumoral alfa/genética
13.
Methods Mol Biol ; 2262: 349-360, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977489

RESUMO

Human cell line models have been widely used for testing of novel anticancer compounds and for predicting clinical response to monotherapies and combinatorial therapies. For many years, standard monolayer culture conditions were used as gold standard, only surpassed by in vivo testing of mouse models. Recently, the incorporation of three-dimensional culture has been shown to further improve predictive compound testing. In view of the renewed interest in anti-RAS cancer therapy, we provide a protocol for establishing colorectal cancer organoids which are characterized by a high prevalence of KRAS mutations.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Mutação , Técnicas de Cultura de Órgãos/métodos , Organoides/patologia , Proteínas ras/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Imunofluorescência , Humanos , Organoides/metabolismo
14.
J Vis Exp ; (170)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33999032

RESUMO

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.


Assuntos
Adenocarcinoma , Antineoplásicos/farmacologia , Neoplasias Colorretais , Avaliação Pré-Clínica de Medicamentos/métodos , Neoplasias de Cabeça e Pescoço , Organoides/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/radioterapia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/radioterapia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Técnicas de Cultura de Órgãos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia
15.
AAPS PharmSciTech ; 22(4): 150, 2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-33973096

RESUMO

Suspension-based matrix transdermal delivery systems (TDSs) are specialized systems that maintain a continuous driving force for drug delivery over prolonged wear. The pressure-sensitive adhesive (PSA) is the most critical constituent of such systems. Our study aimed to determine the effect of different mixing methods on the performance of silicone PSA-based suspension TDSs. Lidocaine suspension TDSs were prepared using conventional slow rotary mixing, high-speed homogenization, bead-mill homogenization, vortex shaking, and by an unguator. Resultant TDSs were tested for tack, shear, and peel properties and correlated to coat weight, content uniformity, microstructure, and in vitro permeation across dermatomed human skin. Every mixing method tested caused a significant reduction in peel. However, bead-mill homogenization resulted in significant loss of all adhesive properties tested, while unguator-mixed TDSs retained most properties. Good linear correlation (R2 = 1.000) between the shear properties of the TDSs with the average cumulative amount of lidocaine permeated after 24 h was observed, with no significant difference between percutaneous delivery from slow rotary-mixed systems (1334 ± 59.21 µg/cm2) and unguator-mixed systems (1147 ± 108.3 µg/cm2). However, significantly lower delivery from bead-mill homogenized systems (821.1 ± 28.00 µg/cm2) was noted. While many factors affect TDS performance, careful consideration must also be given to the processing parameters during development as they have been shown to affect the resultant system's therapeutic efficacy. Extensive mixing with bead-mill homogenization demonstrated crystallization of drug, loss in adhesive properties, coat weight, and film thickness, with reduced transdermal delivery of lidocaine from the prepared system.


Assuntos
Adesivos/administração & dosagem , Adesivos/síntese química , Sistemas de Liberação de Medicamentos/métodos , Absorção Cutânea/efeitos dos fármacos , Adesivo Transdérmico , Adesivos/farmacocinética , Administração Cutânea , Anestésicos Locais/administração & dosagem , Anestésicos Locais/síntese química , Anestésicos Locais/farmacocinética , Humanos , Lidocaína/administração & dosagem , Lidocaína/síntese química , Lidocaína/farmacocinética , Óleo Mineral/administração & dosagem , Óleo Mineral/síntese química , Óleo Mineral/farmacocinética , Técnicas de Cultura de Órgãos , Silicones/metabolismo , Silicones/farmacologia , Absorção Cutânea/fisiologia , Suspensões
16.
Toxicol Appl Pharmacol ; 423: 115570, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33965372

RESUMO

The expression of transporters on the apical and basal membranes of renal proximal tubular cells are down- or upregulated to various extents under cisplatin (CDDP)-induced acute kidney injury (AKI). However, little is known about the changes in transporters in tissues other than the kidney under CDDP-induced AKI. This study aimed to investigate the modulation of the expression/function of intestinal efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), in CDDP-induced AKI rats. On day 3 after the intraperitoneal administration of CDDP (5 mg/kg) to rats, the expression levels of P-gp and Bcrp were compared with those of normal rats. Further, the absorption of three P-gp substrates (6α-methylprednisolone, rhodamine 123, and gatifloxacin) was evaluated in both groups using conventional loop techniques. In the CDDP-induced AKI rats, P-gp expression in the ileum was markedly decreased to approximately 38% of that in the normal rats. However, no significant changes in Bcrp expression were observed in the AKI rats. In contrast with the reduction in P-gp expression in the AKI rats, the absorption of the three P-gp substrates remained almost the same or decreased in the AKI group. The addition of verapamil (a potent P-gp inhibitor) increased the absorption of the three P-gp substrates to the values obtained from the normal rats. In conclusion, our results suggested that P-gp expression is downregulated in rats with CDDP-induced AKI but that P-gp maintains its potency as a "gatekeeper" against the absorption of xenobiotics by amplifying its individual transport capacity under these conditions.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Cisplatino/toxicidade , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/biossíntese , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Injúria Renal Aguda/genética , Animais , Antineoplásicos/toxicidade , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Expressão Gênica , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley
17.
Biochem Pharmacol ; 188: 114560, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33844984

RESUMO

Emerging evidence suggests that G protein coupled receptor 55 (GPR55) may influence adrenoceptor function/activity in the cardiovascular system. Whether this reflects direct interaction (dimerization) between receptors or signalling crosstalk has not been investigated. This study explored the interaction between GPR55 and the alpha 1A-adrenoceptor (α1A-AR) in the cardiovascular system and the potential to influence function/signalling activities. GPR55 and α1A-AR mediated changes in both cardiac and vascular function was assessed in male wild-type (WT) and GPR55 homozygous knockout (GPR55-/-) mice by pressure volume loop analysis and isolated vessel myography, respectively. Dimerization of GPR55 with the α1A-AR was examined in transfected Chinese hamster ovary-K1 (CHO-K1) cells via Bioluminescence Resonance Energy Transfer (BRET). GPR55 and α1A-AR mediated signalling (extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation) was investigated in neonatal rat ventricular cardiomyocytes using AlphaScreen proximity assays. GPR55-/- mice exhibited both enhanced pressor and inotropic responses to A61603 (α1A-AR agonist), while in isolated vessels, A61603 induced vasoconstriction was attenuated by a GPR55-dependent mechanism. Conversely, GPR55-mediated vasorelaxation was not altered by pharmacological blockade of α1A-ARs with tamsulosin. While cellular studies demonstrated that GPR55 and α1A-AR failed to dimerize, pharmacological blockade of GPR55 altered α1A-AR mediated signalling and reduced ERK1/2 phosphorylation. Taken together, this study provides evidence that GPR55 and α1A-AR do not dimerize to form heteromers, but do interact at the signalling level to modulate the function of α1A-AR in the cardiovascular system.


Assuntos
Multimerização Proteica/fisiologia , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Receptores de Canabinoides/deficiência , Receptores de Canabinoides/genética , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Animais Recém-Nascidos , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Gravidez , Multimerização Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
18.
Psychopharmacology (Berl) ; 238(8): 2105-2120, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33837810

RESUMO

BACKGROUND: Depression causes significant debilitating symptoms and economic burden. Current management is challenged by slow onset of action and modest efficacies of antidepressants; thus, the search for newer antidepressants remains relevant. We evaluated the antidepressant effects of a kaurene diterpene, xylopic acid (XA), in zebrafish and mouse models. METHODS: The chronic unpredictable stress (CUS) protocol in zebrafish and the tail suspension test (TST), forced swim test (FST), lipopolysaccharide-induced depression-like behaviour test (LID) and repeated open space swimming test (OSST) in mice were used. We further examined the impact of depleting monoamines on XA's antidepressant effects. The contribution of glutamatergic and nitrergic pathways on the antidepressant effect of XA in mice and XA's effects on 5-HT receptors and monoamine oxidase (MAO) enzymes were also evaluated. Finally, XA's influence on neuroprotection was evaluated by measuring BDNF and oxidative stress enzymes in whole brain. XA doses (1-10 µM) in zebrafish and (10, 30, 100 mg kg-1) in mice exerted potent antidepressant-like potential in FST, TST, LID and showed fast-onset antidepressant-like property in the OSST. RESULTS: The antidepressant-like properties in mice were reversed by blocking synthesis/release of serotonin but not noradrenaline using p-chlorophenylalanine and α-methyl-p-tyrosine, respectively. This antidepressant-like effect was potentiated by D-cycloserine and Nω-Nitro-L-arginine methyl ester (L-NAME) but not by D-serine and L-arginine. XA also evoked partial agonist-like effects on 5-hydroxytrptamine receptors on the rat fundus but it did not have MAO inhibition effect. It also increased BDNF, glutathione and antioxidant enzymes. CONCLUSION: Therefore, xylopic acid possesses antidepressant-like effects largely mediated by serotonergic and neuroprotective mechanisms.


Assuntos
Antidepressivos/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Depressão/tratamento farmacológico , Diterpenos do Tipo Caurano/uso terapêutico , Serotonina/metabolismo , Animais , Antidepressivos/farmacologia , Depressão/metabolismo , Depressão/psicologia , Diterpenos do Tipo Caurano/farmacologia , Relação Dose-Resposta a Droga , Elevação dos Membros Posteriores/efeitos adversos , Elevação dos Membros Posteriores/psicologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Antagonistas da Serotonina/farmacologia , Antagonistas da Serotonina/uso terapêutico , Natação/psicologia , Peixe-Zebra
19.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925529

RESUMO

Mesenchymal stem cell therapy (MSCT) has been shown to be a new therapeutic option for treating alopecia areata (AA). Outer root sheath cells (ORSCs) play key roles in maintaining the hair follicle structure and supporting the bulge area. In human ORSCs (hORSCs), the mechanism for this process has not been extensively studied. In this study, we aimed to examine the influence of human hematopoietic mesenchymal stem cells (hHMSCs) in the hORSCs in vitro model of AA and determine the mechanisms controlling efficacy. Interferon-gamma (IFN-γ) pretreatment was used to induce an in vitro model of AA in hORSCs. The effect of MSCT on the viability and migration of hORSCs was examined using co-cultures, the MTT assay, and migration assays. We investigated the expression of molecules related to the Wnt/ß-catenin pathway, JAK/STAT pathway, and growth factors in hHMSC-treated hORSCs by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. hHMSCs increased hORSC viability and migration when they were co-cultured. hHMSCs reverted IFN-γ-induced expression-including NLRP3, ASC, caspase-1, CXCL-9 through 11, IL-1ß, and IL-15-and upregulated several growth factors and hair stem cell markers. hHMSCs activated several molecules in the Wnt/ß-catenin signaling pathway, such as in the Wnt families, ß-catenin, phosphorylated GSK-3ß and cyclin D1, and suppressed the expression of DKK1 induced by IFN-γ in hORSCs. hHMSCs suppressed the phosphorylation of JAK1 to 3, STAT1, and STAT3 compared to the controls and IFN-γ-pretreated hORSCs. These results demonstrate that hHMSCs increased hORSC viability and migration in the in vitro AA model. Additionally, MSCT definitely stimulated anagen survival and hair growth in an HF organ culture model. MSCT appeared to be associated with the Wnt/ß-catenin and JAK/STAT pathways in hORSCs.


Assuntos
Folículo Piloso/citologia , Folículo Piloso/crescimento & desenvolvimento , Interferon gama/farmacologia , Células-Tronco Mesenquimais/metabolismo , Alopecia em Áreas/metabolismo , Alopecia em Áreas/patologia , Animais , Movimento Celular , Técnicas de Cocultura , Dermatite/metabolismo , Feminino , Expressão Gênica , Folículo Piloso/metabolismo , Humanos , Interferon gama/metabolismo , Janus Quinases/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Técnicas de Cultura de Órgãos , Fatores de Transcrição STAT/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
20.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33805154

RESUMO

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal-epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.


Assuntos
Autoantígenos/metabolismo , Epidermólise Bolhosa Juncional/genética , Colágenos não Fibrilares/metabolismo , Oligonucleotídeos Antissenso/genética , Splicing de RNA , Processamento Alternativo , Biópsia , Linhagem Celular , Sobrevivência Celular , Epidermólise Bolhosa Juncional/metabolismo , Epidermólise Bolhosa Juncional/terapia , Éxons , Genótipo , Homozigoto , Humanos , Queratinócitos/citologia , Lipossomos/química , Mutação , Técnicas de Cultura de Órgãos , RNA Mensageiro/metabolismo
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