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1.
Phys Rev Lett ; 125(8): 088102, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32909763

RESUMO

We perform a bidimensional Stokes experiment in an active cellular material: an autonomously migrating monolayer of Madin-Darby canine kidney epithelial cells flows around a circular obstacle within a long and narrow channel, involving an interplay between cell shape changes and neighbor rearrangements. Based on image analysis of tissue flow and coarse-grained cell anisotropy, we determine the tissue strain rate, cell deformation, and rearrangement rate fields, which are spatially heterogeneous. We find that the cell deformation and rearrangement rate fields correlate strongly, which is compatible with a Maxwell viscoelastic liquid behavior (and not with a Kelvin-Voigt viscoelastic solid behavior). The value of the associated relaxation time is measured as τ=70±15 min, is observed to be independent of obstacle size and division rate, and is increased by inhibiting myosin activity. In this experiment, the monolayer behaves as a flowing material with a Weissenberg number close to one which shows that both elastic and viscous effects can have comparable contributions in the process of collective cell migration.


Assuntos
Movimento Celular/fisiologia , Células Epiteliais/química , Células Epiteliais/citologia , Modelos Biológicos , Substâncias Viscoelásticas/química , Animais , Cães , Células Madin Darby de Rim Canino
2.
Int J Mol Sci ; 21(15)2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32752138

RESUMO

The COVID-19 pandemic caused by the SARS-CoV-2 virus, overlaps with the ongoing epidemics of cigarette smoking and electronic cigarette (e-cig) vaping. However, there is scarce data relating COVID-19 risks and outcome with cigarette or e-cig use. In this study, we mined three independent RNA expression datasets from smokers and vapers to understand the potential relationship between vaping/smoking and the dysregulation of key genes and pathways related to COVID-19. We found that smoking, but not vaping, upregulates ACE2, the cellular receptor that SARS-CoV-2 requires for infection. Both smoking and use of nicotine and flavor-containing e-cigs led to upregulation of pro-inflammatory cytokines and inflammasome-related genes. Specifically, chemokines including CCL20 and CXCL8 are upregulated in smokers, and CCL5 and CCR1 are upregulated in flavor/nicotine-containing e-cig users. We also found genes implicated in inflammasomes, such as CXCL1, CXCL2, NOD2, and ASC, to be upregulated in smokers and these e-cig users. Vaping flavor and nicotine-less e-cigs, however, did not lead to significant cytokine dysregulation and inflammasome activation. Release of inflammasome products, such as IL-1B, and cytokine storms are hallmarks of COVID-19 infection, especially in severe cases. Therefore, our findings demonstrated that smoking or vaping may critically exacerbate COVID-19-related inflammation or increase susceptibility to COVID-19.


Assuntos
Sistemas Eletrônicos de Liberação de Nicotina , Sistema Imunitário/metabolismo , Peptidil Dipeptidase A/metabolismo , Fumar Tabaco , Adulto , Betacoronavirus/isolamento & purificação , Brônquios/citologia , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Pessoa de Meia-Idade , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Regulação para Cima , Adulto Jovem
3.
Methods Mol Biol ; 2203: 119-134, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833209

RESUMO

Well-differentiated primary airway epithelial cell (AEC) cultures have been widely used for the characterization of several human respiratory viruses including coronaviruses. In recent years, there has been an increase in interest toward animal AEC cultures and their application to characterize veterinary viruses with zoonotic potential, as well as studying host-pathogen interactions in animal reservoir host species. In this chapter, we provide a revised and improved protocol for the isolation and establishment of well-differentiated AEC cultures from diverse mammalian species and the use of the cultures for the characterization of veterinary coronavirus. We also describe immunohistochemistry protocols with validated antibodies for the visualization and identification of viral cell tropism in well-differentiated AEC cultures from human, swine, bovine, and feline origin.


Assuntos
Brônquios/citologia , Coronavirus/fisiologia , Células Epiteliais/virologia , Cultura Primária de Células/métodos , Traqueia/citologia , Animais , Gatos , Bovinos , Diferenciação Celular , Células Epiteliais/citologia , Imunofluorescência , Humanos , Imuno-Histoquímica/métodos , Cultura Primária de Células/instrumentação , Suínos , Tropismo Viral
4.
Int J Nanomedicine ; 15: 5043-5060, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764935

RESUMO

Background: Hydroxyapatite (HAP) is a common component of most idiopathic calcium oxalate (CaOx) stones and is often used as a nidus to induce the formation of CaOx kidney stones. Methods: This work comparatively studies the cytotoxicity of four kinds of HAP crystals with different sizes (40 nm to 2 µm), namely, HAP-40 nm, HAP-70 nm, HAP-1 µm, and HAP-2 µm, on human renal proximal tubular epithelial cells (HK-2). Results: HAP crystals reduce the viability and membrane integrity of HK-2 cells in a concentration-dependent manner and consequently cause cytoskeleton damage, cell swelling, increased intracellular reactive oxygen species level, decreased mitochondrial membrane potential, increased intracellular calcium concentration, blocked cell cycle and stagnation in G0/G1 phase, and increased cell necrosis rate. HAP toxicity to HK-2 cells increases with a decrease in crystal size. Conclusion: Cell damage caused by HAP crystals increases the risk of kidney stone formation.


Assuntos
Citotoxinas/química , Citotoxinas/toxicidade , Durapatita/química , Durapatita/toxicidade , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Oxalato de Cálcio/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
5.
PLoS One ; 15(8): e0237766, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822364

RESUMO

Semen contains epithelial cells that can be cultured in vitro. For somatic cell nuclear transfer applications, it is essential to know whether clone(s) produced from semen-derived epithelial cells (SedECs) are healthy and reproductively competent. In this study, the semen and fertility profile of a cloned bull (C1) that was produced from a SedEC were compared with its donor (D1) and with two cloned bulls (C2, C3) that were produced from commonly used skin-derived fibroblast cells (SkdFCs). We observed variations in some fresh semen parameters (ejaculated volume and mass motility), frozen-thawed sperm parameters (plasma membrane integrity, and computer-assisted semen analysis (CASA) indices), but values are within the normal expected range. There was no difference in sperm concentration of ejaculated semen and frozen-thawed semen parameters which include sperm motility, percentage of live and normal morphology sperm, and distance traveled through oestrus mucus. Following in vitro fertilization (IVF) experiments, zygotes from C1 had higher (P < 0.05) cleavage rates (81%) than C2, C3, and D1 (71%, 67%, and 75%, respectively); however, blastocyst development per cleaved embryo and quality of produced blastocysts did not differ. The conception rate of C1 was 46% (7/15) and C2 was 50% (8/15) following artificial insemination with frozen-thawed semen. Established pregnancies resulted in births of 7 and 6 progenies sired by C1 and C2, respectively, and all calves show no signs of phenotypical abnormalities. These results showed that semen from a cloned bull derived from SedECs is equivalent to semen from its donor bull and bulls cloned from SkdFCs.


Assuntos
Búfalos/fisiologia , Clonagem de Organismos/veterinária , Células Epiteliais/citologia , Sêmen/citologia , Animais , Criopreservação/veterinária , Feminino , Fertilidade , Fertilização , Fertilização In Vitro/veterinária , Inseminação Artificial/veterinária , Masculino , Análise do Sêmen/veterinária , Motilidade Espermática
6.
PLoS One ; 15(8): e0237885, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32853234

RESUMO

Our group has developed two transplantation models for the engraftment of Human Intestinal Organoids (HIOs): the renal subcapsular space (RSS) and the mesentery each with specific benefits for study. While engraftment at both sites generates laminated intestinal structures, a direct comparison between models has not yet been performed. Embryonic stem cells were differentiated into HIOs, as previously described. HIOs from the same batch were transplanted on the same day into either the RSS or mesentery. 10 weeks were allowed for engraftment and differentiation, at which time they were harvested and assessed. Metrics for comparison included: mortality, engraftment rate, gross size, number and grade of lumens, and expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism. Mortality was significantly increased when undergoing mesentery transplantation, however engraftment was significantly higher. Graft sizes were similar between groups. Morphometric parameters were similar between groups, however m-tHIOs presented with significantly fewer lumens than k-tHIO. Transcript and protein level expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism were similar between groups. Transplantation into both sites yields viable tissue of similar quality based on our assessments with enhanced engraftment and a dominant lumen for uniform study benefiting the mesenteric site and survival benefiting RSS.


Assuntos
Intestinos/transplante , Organoides/transplante , Animais , Metabolismo dos Carboidratos , Linhagem da Célula , Células Epiteliais/citologia , Sobrevivência de Enxerto , Humanos , Masculino , Camundongos Endogâmicos NOD , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Phys Rev Lett ; 125(3): 038003, 2020 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-32745423

RESUMO

Experiments and theory have shown that cell monolayers and epithelial tissues exhibit solid-liquid and glass-liquid transitions. These transitions are biologically relevant to our understanding of embryonic development, wound healing, and cancer. Current models of confluent epithelia have focused on the role of cell shape, with less attention paid to cell extrusion, which is key for maintaining homeostasis in biological tissue. Here, we use a multiphase field model to study the solid-liquid transition in a confluent monolayer of deformable cells. Cell overlap is allowed and provides a way for modeling the precursor for extrusion. When cells overlap rather than deform, we find that the melting transition changes from continuous to first order like, and that there is an intermittent regime close to the transition, where solid and liquid states alternate over time. By studying the dynamics of five- and sevenfold disclinations in the hexagonal lattice formed by the cell centers, we observe that these correlate with spatial fluctuations in the cellular overlap, and that cell extrusion tends to initiate near fivefold disclinations.


Assuntos
Células Epiteliais/química , Células Epiteliais/citologia , Rim/química , Rim/citologia , Modelos Biológicos , Animais , Fenômenos Biofísicos , Movimento Celular/fisiologia , Forma Celular/fisiologia , Cães , Transição Epitelial-Mesenquimal , Células Madin Darby de Rim Canino , Transição de Fase
8.
Nat Commun ; 11(1): 3805, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732886

RESUMO

The study of organoids, artificially grown cell aggregates with the functionality and small-scale anatomy of real organs, is one of the most active areas of research in biology and biophysics, yet the basic physical origins of their different morphologies remain poorly understood. Here, we propose a mechanistic theory of epithelial shells which resemble small-organoid morphologies. Using a 3D surface tension-based vertex model, we reproduce the characteristic shapes from branched and budded to invaginated structures. We find that the formation of branched morphologies relies strongly on junctional activity, enabling temporary aggregations of topological defects in cell packing. To elucidate our numerical results, we develop an effective elasticity theory, which allows one to estimate the apico-basal polarity from the tissue-scale modulation of cell height. Our work provides a generic interpretation of the observed epithelial shell morphologies, highlighting the role of physical factors such as differential surface tension, cell rearrangements, and tissue growth.


Assuntos
Forma Celular/fisiologia , Células Epiteliais/citologia , Organoides/citologia , Organoides/crescimento & desenvolvimento , Animais , Fenômenos Biomecânicos , Proliferação de Células/fisiologia , Simulação por Computador , Modelos Biológicos , Tensão Superficial
9.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32629995

RESUMO

Peptidylarginine deiminases (PADs) are a family of calcium-regulated enzymes that are phylogenetically conserved and cause post-translational deimination/citrullination, contributing to protein moonlighting in health and disease. PADs are implicated in a range of inflammatory and autoimmune conditions, in the regulation of extracellular vesicle (EV) release, and their roles in infection and immunomodulation are known to some extent, including in viral infections. In the current study we describe putative roles for PADs in COVID-19, based on in silico analysis of BioProject transcriptome data (PRJNA615032 BioProject), including lung biopsies from healthy volunteers and SARS-CoV-2-infected patients, as well as SARS-CoV-2-infected, and mock human bronchial epithelial NHBE and adenocarcinoma alveolar basal epithelial A549 cell lines. In addition, BioProject Data PRJNA631753, analysing patients tissue biopsy data (n = 5), was utilised. We report a high individual variation observed for all PADI isozymes in the patients' tissue biopsies, including lung, in response to SARS-CoV-2 infection, while PADI2 and PADI4 mRNA showed most variability in lung tissue specifically. The other tissues assessed were heart, kidney, marrow, bowel, jejunum, skin and fat, which all varied with respect to mRNA levels for the different PADI isozymes. In vitro lung epithelial and adenocarcinoma alveolar cell models revealed that PADI1, PADI2 and PADI4 mRNA levels were elevated, but PADI3 and PADI6 mRNA levels were reduced in SARS-CoV-2-infected NHBE cells. In A549 cells, PADI2 mRNA was elevated, PADI3 and PADI6 mRNA was downregulated, and no effect was observed on the PADI4 or PADI6 mRNA levels in infected cells, compared with control mock cells. Our findings indicate a link between PADI expression changes, including modulation of PADI2 and PADI4, particularly in lung tissue, in response to SARS-CoV-2 infection. PADI isozyme 1-6 expression in other organ biopsies also reveals putative links to COVID-19 symptoms, including vascular, cardiac and cutaneous responses, kidney injury and stroke. KEGG and GO pathway analysis furthermore identified links between PADs and inflammatory pathways, in particular between PAD4 and viral infections, as well as identifying links for PADs with a range of comorbidities. The analysis presented here highlights roles for PADs in-host responses to SARS-CoV-2, and their potential as therapeutic targets in COVID-19.


Assuntos
Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Desiminases de Arginina em Proteínas/metabolismo , Betacoronavirus/isolamento & purificação , Estudos de Casos e Controles , Linhagem Celular , Infecções por Coronavirus/metabolismo , Citocinas/metabolismo , Bases de Dados Factuais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Vesículas Extracelulares/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Pulmão/enzimologia , Pulmão/patologia , Pulmão/virologia , Pandemias , Pneumonia Viral/metabolismo , Mapas de Interação de Proteínas , Desiminases de Arginina em Proteínas/genética , RNA Mensageiro/metabolismo
10.
Food Chem ; 333: 127509, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32717715

RESUMO

Due to their antioxidant properties, polyphenols are finding novel applications in the fields of Food Technology and Functional Nutrition in the development of innovative functional food products, and in Cosmetics and Regenerative Medicine in the development of formulations for skin disorders. The added-value of polyphenols in these areas is intimately linked to the health benefits they induce which in turn is related to the permeability across the epithelial membrane, a parameter obtained through biomimetic models. This work overviews the knowledge on the interactions of polyphenols with membrane lipids in in vitro models and the underlying challenges in translating biophysical changes achieved with current oversimplified membrane models highlighting the need for improved epithelial membrane models and in turn a better knowledge of the epithelial lipidome. Improved insight into the polyphenol-lipid interactions in vivo (patho)physiological processes will open new opportunities for the exploitation of Food and Agro-Food waste products in Health-related areas.


Assuntos
Lipidômica , Lipídeos/química , Lipídeos de Membrana/química , Polifenóis/química , Animais , Biomimética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Fluidez de Membrana
11.
Respir Res ; 21(1): 182, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32664949

RESUMO

BACKGROUND: Severe acute respiratory syndrome (SARS)-CoV-2-induced coronavirus disease-2019 (COVID-19) is a pandemic disease that affects > 2.8 million people worldwide, with numbers increasing dramatically daily. However, there is no specific treatment for COVID-19 and much remains unknown about this disease. Angiotensin-converting enzyme (ACE)2 is a cellular receptor of SARS-CoV-2. It is cleaved by type II transmembrane serine protease (TMPRSS)2 and disintegrin and metallopeptidase domain (ADAM)17 to assist viral entry into host cells. Clinically, SARS-CoV-2 infection may result in acute lung injury and lung fibrosis, but the underlying mechanisms of COVID-19 induced lung fibrosis are not fully understood. METHODS: The networks of ACE2 and its interacting molecules were identified using bioinformatic methods. Their gene and protein expressions were measured in human epithelial cells after 24 h SARS-CoV-2 infection, or in existing datasets of lung fibrosis patients. RESULTS: We confirmed the binding of SARS-CoV-2 and ACE2 by bioinformatic analysis. TMPRSS2, ADAM17, tissue inhibitor of metalloproteinase (TIMP)3, angiotensinogen (AGT), transformation growth factor beta (TGFB1), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) A and fibronectin (FN) were interacted with ACE2, and the mRNA and protein of these molecules were expressed in lung epithelial cells. SARS-CoV-2 infection increased ACE2, TGFB1, CTGF and FN1 mRNA that were drivers of lung fibrosis. These changes were also found in lung tissues from lung fibrosis patients. CONCLUSIONS: Therefore, SARS-CoV-2 binds with ACE2 and activates fibrosis-related genes and processes to induce lung fibrosis.


Assuntos
Infecções por Coronavirus/genética , Regulação da Expressão Gênica , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Fibrose Pulmonar/genética , Síndrome do Desconforto Respiratório do Adulto/genética , Vírus da SARS/genética , China , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/fisiopatologia , Progressão da Doença , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Pandemias/estatística & dados numéricos , Pneumonia Viral/epidemiologia , Pneumonia Viral/fisiopatologia , Prevalência , Fibrose Pulmonar/epidemiologia , Fibrose Pulmonar/etiologia , Receptores Virais/metabolismo , Síndrome do Desconforto Respiratório do Adulto/diagnóstico , Síndrome do Desconforto Respiratório do Adulto/epidemiologia , Medição de Risco , Análise de Sobrevida , Transcrição Genética , Ativação Transcricional/genética
12.
Prostate ; 80(13): 1145-1156, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32659025

RESUMO

BACKGROUND: Epithelial stem cells (ESCs) demonstrate a capacity to maintain normal tissues homeostasis and ESCs with a deregulated behavior can contribute to cancer development. The ability to reprogram normal tissue epithelial cells into prostate or mammary stem-like cells holds great promise to help understand cell of origin and lineage plasticity in prostate and breast cancers in addition to understanding normal gland development. We previously showed that an intracellular chemokine, CXCL12γ induced cancer stem cells and neuroendocrine characteristics in both prostate and breast adenocarcinoma cell lines. However, its role in normal prostate or mammary epithelial cell fate and development remains unknown. Therefore, we sought to elucidate the functional role of CXCL12γ in the regulation of ESCs and tissue development. METHODS: Prostate epithelial cells (PNT2) or mammary epithelial cells (MCF10A) with overexpressed CXCL12γ was characterized by quantitative real-time polymerase chain reaction, Western blots, and immunofluorescence for lineage marker expression, and fluorescence activated cell sorting analyses and sphere formation assays to examine stem cell surface phenotype and function. Xenotransplantation animal models were used to evaluate gland or acini formation in vivo. RESULTS: Overexpression of CXCL12γ promotes the reprogramming of cells with a differentiated luminal phenotype to a nonluminal phenotype in both prostate (PNT2) and mammary (MCF10A) epithelial cells. The CXCL12γ-mediated nonluminal type cells results in an increase of epithelial stem-like phenotype including the subpopulation of EPCAMLo /CD49fHi /CD24Lo /CD44Hi cells capable of sphere formation. Critically, overexpression of CXCL12γ promotes the generation of robust gland-like structures from both prostate and mammary epithelial cells in in vivo xenograft animal models. CONCLUSIONS: CXCL12γ supports the reprogramming of epithelial cells into nonluminal cell-derived stem cells, which facilitates gland development.


Assuntos
Quimiocina CXCL12/biossíntese , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Próstata/crescimento & desenvolvimento , Animais , Reprogramação Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Xenoenxertos , Humanos , Masculino , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Camundongos , Próstata/citologia , Próstata/metabolismo , Isoformas de Proteínas
13.
Postepy Biochem ; 66(2): 151-159, 2020 06 27.
Artigo em Polonês | MEDLINE | ID: mdl-32700509

RESUMO

The epithelial tissues have continuous contact with external environment, including pathogenic microorganisms. Endogenous antimicrobial proteins and peptides produced by epithelial cells play a key role in controlling microbial burden and composition, either directly, or by engaging immune cells. These include active derivatives of multifunctional protein chemerin, which is equipped with both antimicrobial and chemotactic function. Given an increasing number of infections caused by antibiotic-insensitive microorganisms, such as methicillin- resistant S. aureus (MRSA), it is important to fully understand how these epithelia-associated microorganisms are controlled at barrier sites, including skin and oral cavity. Chemerin-derived synthetic peptide 4 (p4) covering central Val66-Pro85 chemerin sequence exhibits broad range of antimicrobial activity against skin- and oral cavity- associated bacteria, including MRSA strains, suggesting its therapeutic potential for bacteria-mediated barrier organs pathologies. In this article we present the overview of protective functions of chemerin and chemerin-derived peptides in the epithelial tissues.


Assuntos
Antibacterianos/metabolismo , Bactérias/metabolismo , Quimiocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Farmacorresistência Bacteriana , Células Epiteliais/citologia , Humanos , Staphylococcus aureus Resistente à Meticilina/metabolismo
14.
Genes (Basel) ; 11(7)2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32646047

RESUMO

The global spread of COVID-19, caused by pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for an imminent response from medical research communities to better understand this rapidly spreading infection. Employing multiple bioinformatics and computational pipelines on transcriptome data from primary normal human bronchial epithelial cells (NHBE) during SARS-CoV-2 infection revealed activation of several mechanistic networks, including those involved in immunoglobulin G (IgG) and interferon lambda (IFNL) in host cells. Induction of acute inflammatory response and activation of tumor necrosis factor (TNF) was prominent in SARS-CoV-2 infected NHBE cells. Additionally, disease and functional analysis employing ingenuity pathway analysis (IPA) revealed activation of functional categories related to cell death, while those associated with viral infection and replication were suppressed. Several interferon (IFN) responsive gene targets (IRF9, IFIT1, IFIT2, IFIT3, IFITM1, MX1, OAS2, OAS3, IFI44 and IFI44L) were highly upregulated in SARS-CoV-2 infected NBHE cell, implying activation of antiviral IFN innate response. Gene ontology and functional annotation of differently expressed genes in patient lung tissues with COVID-19 revealed activation of antiviral response as the hallmark. Mechanistic network analysis in IPA identified 14 common activated, and 9 common suppressed networks in patient tissue, as well as in the NHBE cell model, suggesting a plausible role for these upstream regulator networks in the pathogenesis of COVID-19. Our data revealed expression of several viral proteins in vitro and in patient-derived tissue, while several host-derived long noncoding RNAs (lncRNAs) were identified. Our data highlights activation of IFN response as the main hallmark associated with SARS-CoV-2 infection in vitro and in human, and identified several differentially expressed lncRNAs during the course of infection, which could serve as disease biomarkers, while their precise role in the host response to SARS-CoV-2 remains to be investigated.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , RNA Longo não Codificante/metabolismo , Proteínas Virais/metabolismo , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Biomarcadores/metabolismo , Brônquios/citologia , Morte Celular , Linhagem Celular , Análise por Conglomerados , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Células Epiteliais/citologia , Células Epiteliais/virologia , Redes Reguladoras de Genes , Humanos , Imunidade Inata , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Pandemias , Pneumonia Viral/genética , Pneumonia Viral/virologia , RNA Longo não Codificante/genética , Transcriptoma
15.
Nature ; 583(7815): 296-302, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612232

RESUMO

The mammalian immune system implements a remarkably effective set of mechanisms for fighting pathogens1. Its main components are haematopoietic immune cells, including myeloid cells that control innate immunity, and lymphoid cells that constitute adaptive immunity2. However, immune functions are not unique to haematopoietic cells, and many other cell types display basic mechanisms of pathogen defence3-5. To advance our understanding of immunology outside the haematopoietic system, here we systematically investigate the regulation of immune genes in the three major types of structural cells: epithelium, endothelium and fibroblasts. We characterize these cell types across twelve organs in mice, using cellular phenotyping, transcriptome sequencing, chromatin accessibility profiling and epigenome mapping. This comprehensive dataset revealed complex immune gene activity and regulation in structural cells. The observed patterns were highly organ-specific and seem to modulate the extensive interactions between structural cells and haematopoietic immune cells. Moreover, we identified an epigenetically encoded immune potential in structural cells under tissue homeostasis, which was triggered in response to systemic viral infection. This study highlights the prevalence and organ-specific complexity of immune gene activity in non-haematopoietic structural cells, and it provides a high-resolution, multi-omics atlas of the epigenetic and transcriptional networks that regulate structural cells in the mouse.


Assuntos
Endotélio/imunologia , Células Epiteliais/imunologia , Fibroblastos/imunologia , Regulação da Expressão Gênica/imunologia , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Especificidade de Órgãos/imunologia , Imunidade Adaptativa , Animais , Cromatina/genética , Cromatina/metabolismo , Endotélio/citologia , Epigênese Genética/imunologia , Epigenoma/genética , Células Epiteliais/citologia , Feminino , Fibroblastos/citologia , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Sistema Imunitário/virologia , Imunidade Inata , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Especificidade de Órgãos/genética , Transcrição Genética/imunologia , Transcriptoma/genética
16.
Virol Sin ; 35(3): 280-289, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32557270

RESUMO

Cancer cell lines have been used widely in cancer biology, and as biological or functional cell systems in many biomedical research fields. These cells are usually defective for many normal activities or functions due to significant genetic and epigenetic changes. Normal primary cell yields and viability from any original tissue specimens are usually relatively low or highly variable. These normal cells cease after a few passages or population doublings due to very limited proliferative capacity. Animal models (ferret, mouse, etc.) are often used to study virus-host interaction. However, viruses usually need to be adapted to the animals by several passages due to tropism restrictions including viral receptors and intracellular restrictions. Here we summarize applications of conditionally reprogrammed cells (CRCs), long-term cultures of normal airway epithelial cells from human nose to lung generated by conditional cell reprogramming (CR) technology, as an ex vivo model in studies of emerging viruses. CR allows to robustly propagate cells from non-invasive or minimally invasive specimens, for example, nasal or endobronchial brushing. This process is rapid (2 days) and conditional. The CRCs maintain their differentiation potential and lineage functions, and have been used for studies of adenovirus, rhinovirus, respiratory syncytial virus, influenza viruses, parvovirus, and SARS-CoV. The CRCs can be easily used for air-liquid interface (ALI) polarized 3D cultures, and these coupled CRC/ALI cultures mimic physiological conditions and are suitable for studies of viral entry including receptor binding and internalization, innate immune responses, viral replications, and drug discovery as an ex vivo model for emerging viruses.


Assuntos
Técnicas de Reprogramação Celular , Modelos Biológicos , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Betacoronavirus/fisiologia , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Células Epiteliais/citologia , Células Epiteliais/virologia , Humanos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia
17.
J Vis Exp ; (159)2020 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-32478726

RESUMO

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.


Assuntos
Ductos Biliares/citologia , Células Epiteliais/citologia , Animais , Técnicas de Cultura de Células , Polaridade Celular , Cistos , Hidrogéis , Ratos
18.
PLoS Genet ; 16(6): e1008717, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479493

RESUMO

Forces generated by the actomyosin cytoskeleton are key contributors to many morphogenetic processes. The actomyosin cytoskeleton organises in different types of networks depending on intracellular signals and on cell-cell and cell-extracellular matrix (ECM) interactions. However, actomyosin networks are not static and transitions between them have been proposed to drive morphogenesis. Still, little is known about the mechanisms that regulate the dynamics of actomyosin networks during morphogenesis. This work uses the Drosophila follicular epithelium, real-time imaging, laser ablation and quantitative analysis to study the role of integrins on the regulation of basal actomyosin networks organisation and dynamics and the potential contribution of this role to cell shape. We find that elimination of integrins from follicle cells impairs F-actin recruitment to basal medial actomyosin stress fibers. The available F-actin redistributes to the so-called whip-like structures, present at tricellular junctions, and into a new type of actin-rich protrusions that emanate from the basal cortex and project towards the medial region. These F-actin protrusions are dynamic and changes in total protrusion area correlate with periodic cycles of basal myosin accumulation and constriction pulses of the cell membrane. Finally, we find that follicle cells lacking integrin function show increased membrane tension and reduced basal surface. Furthermore, the actin-rich protrusions are responsible for these phenotypes as their elimination in integrin mutant follicle cells rescues both tension and basal surface defects. We thus propose that the role of integrins as regulators of stress fibers plays a key role on controlling epithelial cell shape, as integrin disruption promotes reorganisation into other types of actomyosin networks, in a manner that interferes with proper expansion of epithelial basal surfaces.


Assuntos
Actomiosina/metabolismo , Forma Celular , Proteínas de Drosophila/metabolismo , Células Epiteliais/metabolismo , Integrinas/metabolismo , Fibras de Estresse/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila , Células Epiteliais/citologia , Fibras de Estresse/ultraestrutura
19.
PLoS One ; 15(6): e0235118, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579601

RESUMO

During diabetes, renal proximal tubular cells (PTC) are exposed to a combination of high glucose and hypoxic conditions, which plays a relevant role in the development of diabetic kidney disease (DKD). In this work, a time-series proteomic study was performed to analyse the effect of a diabetic-like microenvironment induced changes on HK-2 cells, a human cell line derived from normal proximal tubular epithelial cells. Cells simultaneously exposed to high glucose (25 mM) and hypoxia (1% O2) were compared to cells in control conditions for up to 48 h. Diabetic conditions increased the percentage of death cells after 24 and 48 h, but no differences in the protein/cell ratio were found. The relative protein quantification using dimethyl-labeling and UHPLC-MS/MS analysis allowed the identification of 317, 296 and 259 proteins at 5, 24 and 48 h, respectively. The combination of statistical and time expression profile analyses indicated an increased expression of proteins involved in glycolysis, and a decrease of cytoskeletal-related proteins. The exposure of HK-2 cells to high glucose and hypoxia reproduces some of the effects of diabetes on PTC and, with the limitations inherent to in vitro studies, propose new mechanisms and targets to be considered in the management of DKD.


Assuntos
Células Epiteliais/metabolismo , Glucose/metabolismo , Túbulos Renais Proximais/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Hipóxia Celular , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Nefropatias Diabéticas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucose/farmacologia , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Espectrometria de Massas em Tandem/métodos , Fatores de Tempo
20.
Prostate ; 80(11): 872-884, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32497356

RESUMO

BACKGROUND: Castration-insensitive epithelial progenitors capable of regenerating the prostate have been proposed to be concentrated in the proximal region based on facultative assays. Functional characterization of prostate epithelial populations isolated with individual cell surface markers has failed to provide a consensus on the anatomical and transcriptional identity of proximal prostate progenitors. METHODS: Here, we use single-cell RNA sequencing to obtain a complete transcriptomic profile of all epithelial cells in the mouse prostate and urethra to objectively identify cellular subtypes. Pan-transcriptomic comparison to human prostate cell types identified a mouse equivalent of human urethral luminal cells, which highly expressed putative prostate progenitor markers. Validation of the urethral luminal cell cluster was performed using immunostaining and flow cytometry. RESULTS: Our data reveal that previously identified facultative progenitors marked by Trop2, Sca-1, KRT4, and PSCA are actually luminal epithelial cells of the urethra that extend into the proximal region of the prostate, and are resistant to castration-induced androgen deprivation. Mouse urethral luminal cells were identified to be the equivalent of previously identified human club and hillock cells that similarly extend into proximal prostate ducts. Benign prostatic hyperplasia (BPH) has long been considered an "embryonic reawakening," but the cellular origin of the hyperplastic growth concentrated in the periurethral region is unclear. We demonstrate an increase in urethral luminal cells within glandular nodules from BPH patients. Urethral luminal cells are further increased in patients treated with a 5-α reductase inhibitor. CONCLUSIONS: Our data demonstrate that cells of the proximal prostate that express putative progenitor markers, and are enriched by castration in the proximal prostate, are urethral luminal cells and that these cells may play an important role in the etiology of human BPH.


Assuntos
Próstata/citologia , Células-Tronco/citologia , Uretra/citologia , Adolescente , Adulto , Animais , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Próstata/metabolismo , Células-Tronco/metabolismo , Uretra/metabolismo , Adulto Jovem
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