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1.
Zhongguo Dang Dai Er Ke Za Zhi ; 22(9): 980-983, 2020 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-32933630

RESUMO

OBJECTIVE: To study the expression level of cAMP response element-binding protein (CREB) in children with recurrent wheezing under three years of age and its effect on the expression of the serum orosomucoid 1-like protein 3 (ORMDL3) gene. METHODS: Thirty-six children with recurrent wheezing under three years of age who visited the hospital from June 2017 to June 2019 were selected as the recurrent wheezing group. Twenty-four healthy children from physical examination were selected as the control group. The CREB expression level in peripheral blood was measured by quantitative real-time PCR. Human bronchial epithelial cells (BEAS-2B) were cultured, and dual-luciferase reporter assay and quantitative real-time PCR were used to investigate the effects of overexpression and siRNA interference of CREB on the promoter activity and mRNA expression of the ORMDL3 gene in the BEAS-2B cells. RESULTS: The expression level of CREB in the recurrent wheezing group was significantly higher than that in the control group (P<0.001). In BEAS-2B cells, overexpression of CREB significantly up-regulated the promoter activity and mRNA expression of the ORMDL3 gene (P<0.05), while siRNA interference of CREB significantly reduced the promoter activity and mRNA expression of the ORMDL3 gene (P<0.05). CONCLUSIONS: The expression of CREB is increased in children with recurrent wheezing, and CREB may be involved in the pathogenesis of recurrent wheezing by regulating expression of the ORMDL3 gene.


Assuntos
Proteínas de Membrana/genética , Sons Respiratórios , Pré-Escolar , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Células Epiteliais , Humanos , Regiões Promotoras Genéticas
2.
Zhongguo Zhong Yao Za Zhi ; 45(16): 3922-3930, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32893590

RESUMO

The aim of this paper was to observe the effect of salvianolic acid B(Sal B) on high-glucose induced renal tubular epithelial-mesenchymal transition(EMT) in rats, and to explore its possible mechanisms of prevention and treatment of diabetic nephropathy. The rat renal tubular epithelial NRK-52 E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+10 µmol·L~(-1)Sal B group(Sal B), the above 3 groups were set at 6, 12, 24 and 48 h for dynamic observation; high glucose+Sal B different concentration(1, 5, 10 µmol·L~(-1)) groups, high glucose+5.0 µmol·L~(-1) pioglitazone group, high glucose+10 µmol·L~(-1)Sal B+5 µmol·L~(-1)GW9662 group. The protein expression levels of PPARγ, PTEN, α-SMA, E-cadherin and PI3 K/Akt signaling molecules were determined by Western blot. The mRNA expression of PPARγ and PTEN were detected by Real-time PCR. The viabi-lity of NRK52 E cells was determined by MTT assay. The results showed that as compared with control group, the mRNA and protein expression levels of PPARγ and PTEN in high glucose group gradually reduced, the protein expression levels of α-SMA and p-Akt~((Thr308))gradually increased, and the protein expression of E-cadherin gradually reduced(P<0.05). As compared with high glucose group, when increases in Sal B doses, the mRNA and protein expression levels of PPARγ, PTEN in high glucose + different concentrations of Sal B groups gradually increased, the protein expression levels of α-SMA and p-Akt~((Thr308)) gradually reduced, and the protein expression of E-cadherin gradually increased(P<0.05), however, the effect of 1 µmol·L~(-1)concentration of Sal B on the expression of PPARγ mRNA and protein and PTEN mRNA was not significantly different. As compared with high glucose group, the mRNA and protein expression levels of PPARγ mRNA(except 6 h) and protein(except 6 h), PTEN mRNA(except 6 h) and protein(except 6, 12 h) kept increasing, the protein expression levels of α-SMA and p-Akt~((Thr308))(except 6 h) continued to reduce, the protein expression of E-cadherin kept increasing in high glucose+10 µmol·L~(-1) Sal B dynamic observation group(P<0.05). As compared with high glucose group, Sal B and the pioglitazone(PIO) can greatly enhance the expression of PPARγ, PTEN at mRNA and protein levels, enhance the expression of E-cadherin at protein levels, and reduce the expression of α-SMA, p-Akt~((Thr308))protein level(P<0.05), there was no significant difference between the two groups. However, the expression levels of PPARγ and PTEN mRNA and protein, E-cadherin, α-SMA and p-Akt(Thr308) protein in the Sal B+GW9662 control group were not statistically significant compared with the high glucose group. The effect of Sal B was blocked by the PPARγ antagonist GW9662. It can be concluded that Sal B can suppress the NRK52 E cells induced by high-glucose EMT. The mechanism may be related to the activation of PPARγ with Sal B, and the up-regulation of PTEN expression, and thereby inhibiting the fibrosis effect of PI3 K/Akt signaling pathway.


Assuntos
Transição Epitelial-Mesenquimal , Fator de Crescimento Transformador beta1 , Animais , Benzofuranos , Células Epiteliais , Glucose , Ratos
3.
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
5.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(4): 469-474, 2020 Apr 30.
Artigo em Chinês | MEDLINE | ID: mdl-32895123

RESUMO

OBJECTIVE: To evaluate the expression of thymidylate synthase (TS) in myoepithelial cells (MECs) of salivary adenoid tissues and explore its clinical significance. METHODS: Immunohistochemical staining EnVision method was used to detect the expression of TS, P63, Calponin, CK5/6 and S-100 in 32 salivary gland specimens, including 10 non-neoplastic and salivary inflammation specimens, 11 mixed tumor specimens, 5 basal cell carcinoma specimens and 6 adenoid cyst carcinoma specimens. The specificity and sensitivity of TS as a specific molecular marker of salivary muscle epithelial cells were evaluated in comparison with P63, Calponin, CK5/6 and S-100. RESULTS: The expression pattern of TS in all the salivary gland tissue specimens was identical with that of p63. TS and P63 both showed strong immunohistochemical expressions in MECs of salivary adenoid tissue specimens. Calponin, CK5/6, and S-100 showed cytoplasmic/membranous expressions in the MECs. In addition, TS exhibited weak or moderate cytoplasmic expression in a few salivary gland epithelial cells, cancer cells and scattered stromal cells, with negative expression in the cell nuclei. The expression of TS in the MECs of all the salivary adenoid specimens was highly consistent with those of P63, Calponin, CK5/6 and S-100 (P>0.05) Except for CK5/6 expression in Salivary inflammation and Salivary gland specimens. Kappa>0.75. The specificity and sensitivity of TS as a molecular marker of MECs were both 100%. CONCLUSIONS: TS is a new specific marker of MECs for differential diagnosis of salivary gland tumors.


Assuntos
Tonsila Faríngea , Células Epiteliais , Biomarcadores Tumorais , Carcinoma Adenoide Cístico , Humanos , Neoplasias das Glândulas Salivares , Timidilato Sintase
6.
Acta Cir Bras ; 35(8): e202000802, 2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32901679

RESUMO

PURPOSE: To investigate the effects of grape seed proanthocyanidin B2 (GSPB2) preconditioning on oxidative stress and apoptosis of renal tubular epithelial cells in mice after renal ischemia-reperfusion (RIR). METHODS: Forty male ICR mice were randomly divided into 4 groups: Group A: mice were treated with right nephrectomy. Group B: right kidney was resected and the left renal vessel was clamped for 45 minutes. Group C: mice were intraperitoneally injected with GSPB2 before RIR established. Group D: mice were intraperitoneally injected with GSPB2 plus brusatol before RIR established. Creatinine and urea nitrogen of mice were determined. Pathological and morphological changes of kidney were checked. Expressions of Nrf-2, HO-1, cleaved-caspase3 were detected by Western-blot. RESULTS: Compared to Group B, morphology and pathological damages of renal tissue were less serious in Group C. Western-blot showed that expressions of Nrf-2 and HO-1 in Group C were obviously higher than those in Group B. The expression of cleaved-caspase3 in Group C was significantly lower than that in Group B. CONCLUSION: GSPB2 preconditioning could attenuate renal oxidative stress injury and renal tubular epithelial cell apoptosis by up-regulating expressions of Nrf-2 and HO-1 and down-regulating the expression of cleaved-caspase-3, but the protective effect could be reversed by brusatol.


Assuntos
Apoptose , Extrato de Sementes de Uva , Estresse Oxidativo , Proantocianidinas , Traumatismo por Reperfusão , Animais , Apoptose/efeitos dos fármacos , Células Epiteliais , Extrato de Sementes de Uva/farmacologia , Extrato de Sementes de Uva/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos ICR , Estresse Oxidativo/efeitos dos fármacos , Proantocianidinas/farmacologia , Proantocianidinas/uso terapêutico
7.
Nat Commun ; 11(1): 3910, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764693

RESUMO

SARS-CoV-2, a ß-coronavirus, has rapidly spread across the world, highlighting its high transmissibility, but the underlying morphogenesis and pathogenesis remain poorly understood. Here, we characterize the replication dynamics, cell tropism and morphogenesis of SARS-CoV-2 in organotypic human airway epithelial (HAE) cultures. SARS-CoV-2 replicates efficiently and infects both ciliated and secretory cells in HAE cultures. In comparison, HCoV-NL63 replicates to lower titers and is only detected in ciliated cells. SARS-CoV-2 shows a similar morphogenetic process as other coronaviruses but causes plaque-like cytopathic effects in HAE cultures. Cell fusion, apoptosis, destruction of epithelium integrity, cilium shrinking and beaded changes are observed in the plaque regions. Taken together, our results provide important insights into SARS-CoV-2 cell tropism, replication and morphogenesis.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Células Epiteliais/virologia , Morfogênese/fisiologia , Pneumonia Viral/virologia , Sistema Respiratório/virologia , Betacoronavirus/patogenicidade , Linhagem Celular , Células Cultivadas , Efeito Citopatogênico Viral , Células Epiteliais/patologia , Humanos , Pandemias , Sistema Respiratório/patologia , Tropismo , Replicação Viral
8.
Zhonghua Yi Xue Za Zhi ; 100(32): 2481-2487, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32829592

RESUMO

Objective: To explore the relationship between family with sequence similarity 13 member A (FAM13A) gene and small airway remodeling in chronic obstructive pulmonary disease (COPD), and the effect of interference with FAM13A gene expression on the apoptosis and proliferation phenotype of human airway epithelial cells (16HBE). Methods: From January 2018 to January 2020, 74 patients in the Department of Thoracic Surgery of General Hospital of Ningxia Medical University were treated by surgery for lung tumors or pulmonary bullae. According to the lung function and smoking history, the 74 patients were divided into four groups: non-smoking group with normal lung function (normal group, 23 patients), smoking group with normal lung function (smoking group, 24 patients), non-smoking group with COPD (11 patients) and smoking group with COPD (16 patients). The expression of FAM13A in small airway of each group was detected by immunohistochemistry, and the correlation between FAM13A and the airflow restriction indexes by pulmonary function was analyzed. The shRNA fragment of FAM13A gene was designed, and the shRNA lentivirus vector of FAM13A gene was constructed and packaged. The expression level of FAM13A gene was detected by real-time fluorescent quantitative PCR (qRT-PCR) and Western blot, and the best shRNA sequence was screened. Flow cytometry was used to detect apoptosis rate and the fluorescence intensity of proliferation marker Ki-67 in 16HBE cells. Results: FAM13A was mainly expressed in the cytoplasm of small airway epithelial cells. The levels of FAM13A absorbance (A) of small airway epithelial cells in non-smoking group and smoking group with COPD were higher than those in normal group and smoking group (0.365±0.026, 0.412±0.053 to 0.113±0.018, 0.105±0.009, all P<0.05), and they were negatively correlated with forced expiratory volume in 1s/forced vital capacity (FEV(1)/FVC) and FEV(1)% pre (r=-0.48 and r=-0.40, all P<0.05). The FAM13A shRNA lentiviral vector was successfully constructed, and FAM13A interference was successfully achieved in the 16HBE cell line. After infection of 16HBE cells, the results of qRT-PCR and Western blot showed that the expression of FAM13A in shRNA-target-2 group decreased (all P<0.01). Compared with the negative control group (shRNA-NC), the apoptosis rate of FAM13A shRNA group decreased (P=0.023), and the fluorescence intensity of Ki-67 also decreased (P=0.042). Conclusions: FAM13A gene expression is increased in COPD small airway epithelial cells, and it is related to COPD airflow limitation. FAM13A gene may participate in the process of COPD remodeling by affecting the apoptosis and proliferation of human airway epithelial cells.


Assuntos
Remodelação das Vias Aéreas , Doença Pulmonar Obstrutiva Crônica , Apoptose , Proliferação de Células , Células Epiteliais , Humanos , Pulmão
9.
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
10.
PLoS Pathog ; 16(8): e1008760, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790753

RESUMO

Influenza A viruses (IAVs) remain a significant global health burden. Activation of the innate immune response is important for controlling early virus replication and spread. It is unclear how early IAV replication events contribute to immune detection. Additionally, while many cell types in the lung can be infected, it is not known if all cell types contribute equally to establish the antiviral state in the host. Here, we use single-cycle influenza A viruses (scIAVs) to characterize the early immune response to IAV in vitro and in vivo. We found that the magnitude of virus replication contributes to antiviral gene expression within infected cells prior to the induction of a global response. We also developed a scIAV that is only capable of undergoing primary transcription, the earliest stage of virus replication. Using this tool, we uncovered replication stage-specific responses in vitro and in vivo. Using several innate immune receptor knockout cell lines, we identify RIG-I as the predominant antiviral detector of primary virus transcription and amplified replication in vitro. Through a Cre-inducible reporter mouse, we used scIAVs expressing Cre-recombinase to characterize cell type-specific responses in vivo. Individual cell types upregulate unique sets of antiviral genes in response to both primary virus transcription and amplified replication. We also identified antiviral genes that are only upregulated in response to direct infection. Altogether, these data offer insight into the early mechanisms of antiviral gene activation during influenza A infection.


Assuntos
Células Epiteliais/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Replicação Viral , Células A549 , Animais , Antivirais/farmacologia , Proteína DEAD-box 58/metabolismo , Cães , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Células HEK293 , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Influenza Humana/tratamento farmacológico , Influenza Humana/patologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia
11.
PLoS Pathog ; 16(8): e1008766, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32857822

RESUMO

Pathogens commonly disrupt the intestinal epithelial barrier; however, how the epithelial immune system senses the loss of intestinal barrier as a danger signal to activate self-defense is unclear. Through an unbiased approach in the model nematode Caenorhabditis elegans, we found that the EGL-44/TEAD transcription factor and its transcriptional activator YAP-1/YAP (Yes-associated protein) were activated when the intestinal barrier was disrupted by infections with the pathogenic bacterium Pseudomonas aeruginosa PA14. Gene Ontology enrichment analysis of the genes containing the TEAD-binding sites revealed that "innate immune response" and "defense response to Gram-negative bacterium" were two top significantly overrepresented terms. Genetic inactivation of yap-1 and egl-44 significantly reduced the survival rate and promoted bacterial accumulation in worms after bacterial infections. Furthermore, we found that disturbance of the E-cadherin-based adherens junction triggered the nuclear translocation and activation of YAP-1/YAP in the gut of worms. Although YAP is a major downstream effector of the Hippo signaling, our study revealed that the activation of YAP-1/YAP was independent of the Hippo pathway during disruption of intestinal barrier. After screening 10 serine/threonine phosphatases, we identified that PP2A phosphatase was involved in the activation of YAP-1/YAP after intestinal barrier loss induced by bacterial infections. Additionally, our study demonstrated that the function of YAP was evolutionarily conserved in mice. Our study highlights how the intestinal epithelium recognizes the loss of the epithelial barrier as a danger signal to deploy defenses against pathogens, uncovering an immune surveillance program in the intestinal epithelium.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Permeabilidade da Membrana Celular , Células Epiteliais/imunologia , Microbioma Gastrointestinal/imunologia , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Camundongos , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Transdução de Sinais
12.
Sci Adv ; 6(33): eabb7238, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32851183

RESUMO

Cigarette smoking, the leading cause of chronic obstructive pulmonary disease (COPD), has been implicated as a risk factor for severe disease in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we show that mice with lung epithelial cell-specific loss of function of Miz1, which we identified as a negative regulator of nuclear factor κB (NF-κB) signaling, spontaneously develop progressive age-related changes resembling COPD. Furthermore, loss of Miz1 up-regulates the expression of Ace2, the receptor for SARS-CoV-2. Concomitant partial loss of NF-κB/RelA prevented the development of COPD-like phenotype in Miz1-deficient mice. Miz1 protein levels are reduced in the lungs from patients with COPD, and in the lungs of mice exposed to chronic cigarette smoke. Our data suggest that Miz1 down-regulation-induced sustained activation of NF-κB-dependent inflammation in the lung epithelium is sufficient to induce progressive lung and airway destruction that recapitulates features of COPD, with implications for COVID-19.


Assuntos
Células Epiteliais/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/metabolismo , Peptidil Dipeptidase A/metabolismo , Fenótipo , Proteínas Inibidoras de STAT Ativados/genética , Doença Pulmonar Obstrutiva Crônica/genética , Ubiquitina-Proteína Ligases/genética , Regulação para Cima/genética , Animais , Betacoronavirus , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Proteínas Inibidoras de STAT Ativados/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transdução de Sinais/genética , Fumar/efeitos adversos , Fator de Transcrição RelA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
13.
Cells ; 9(9)2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32859121

RESUMO

Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells' exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.


Assuntos
Betacoronavirus/química , Infecções por Coronavirus/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Ativação Linfocitária/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Doadores de Sangue , Brônquios/citologia , Degranulação Celular/genética , Técnicas de Cocultura , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Células Epiteliais/metabolismo , Humanos , Interferon gama/metabolismo , Células K562 , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , RNA Viral/genética , Vírus da SARS/química , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Transfecção
14.
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
15.
Adv Exp Med Biol ; 1265: 57-70, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32761570

RESUMO

Lung diseases affect millions of individuals all over the world. Various environmental factors, such as toxins, chemical pollutants, detergents, viruses, bacteria, microbial dysbiosis, and allergens, contribute to the development of respiratory disorders. Exposure to these factors activates stress responses in host cells and disrupt lung homeostasis, therefore leading to dysfunctional epithelial barriers. Despite significant advances in therapeutic treatments for lung diseases in the last two decades, novel interventional targets are imperative, considering the side effects and limited efficacy in patients treated with currently available drugs. Nutrients, such as amino acids (e.g., arginine, glutamine, glycine, proline, taurine, and tryptophan), peptides, and bioactive molecules, have attracted more and more attention due to their abilities to reduce oxidative stress, inhibit apoptosis, and regulate immune responses, thereby improving epithelial barriers. In this review, we summarize recent advances in amino acid metabolism in the lungs, as well as multifaceted functions of amino acids in attenuating inflammatory lung diseases based on data from studies with both human patients and animal models. The underlying mechanisms for the effects of physiological amino acids are likely complex and involve cell signaling, gene expression, and anti-oxidative reactions. The beneficial effects of amino acids are expected to improve the respiratory health and well-being of humans and other animals. Because viruses (e.g., coronavirus) and environmental pollutants (e.g., PM2.5 particles) induce severe damage to the lungs, it is important to determine whether dietary supplementation or intravenous administration of individual functional amino acids (e.g., arginine-HCl, citrulline, N-acetylcysteine, glutamine, glycine, proline and tryptophan) or their combinations to affected subjects may alleviate injury and dysfunction in this vital organ.


Assuntos
Aminoácidos/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Pneumopatias/metabolismo , Pneumopatias/patologia , Animais , Humanos , Pneumopatias/fisiopatologia
16.
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
17.
PLoS One ; 15(7): e0233583, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735619

RESUMO

Mutations that cause Huntington's Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington's disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model. We found mHttex1 did not appear to stall translation of its own nascent chain, or at best was marginal. We also found the inclusions appeared to recruit low levels of RNA but there was no difference in enrichment between early formed and mature inclusions. Proteins involved in translation or ribosome quality control were co-recruited to the inclusions (Ltn1 Rack1) compared to a protein not anticipated to be involved (NACAD), but there was no major specificity of enrichment in the early formed inclusions compared to mature inclusions. Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions also contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation. Collectively our findings show little evidence that inclusion assembly arises by a discrete clustering of stalled nascent chains and associated quality control machinery. Instead, the machinery appear to be recruited continuously, or secondarily, to the nucleation of inclusion formation.


Assuntos
Éxons/genética , Proteína Huntingtina/genética , Elongação Traducional da Cadeia Peptídica , Agregados Proteicos/genética , RNA Mensageiro/genética , Ribossomos/metabolismo , Sequência de Bases , Células Epiteliais , Genes Reporter , Células HEK293 , Humanos , Proteína Huntingtina/biossíntese , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Repetições Minissatélites , Peptídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
18.
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
20.
Ecotoxicol Environ Saf ; 202: 110932, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32800216

RESUMO

Adverse health effects arising from exposure to fine particulates have become a major concern. Angiogenesis is a vital physiological process for the growth and development of cells and structures in the human body, whereby excessive or insufficient vessel growth could contribute to pathogenesis of diseases. We therefore evaluated indirect effects of carbon black (CB) and inhalable airborne particles on the angiogenic ability of unexposed Human Umbilical Vein Endothelial Cells (HUVECs) by co-culturing HUVECs with pre-exposed Small Airway Epithelial Cells (SAECs). As endothelial cells are major components of blood vessels and potential targets of fine particles, we investigated if lung epithelial cells exposed to ambient PM2.5 surrogates could induce bystander effects on neighboring unexposed endothelial cells in an alveolar-capillary co-culture lung model. Epithelial exposure to CB at a non-toxic dose of 25 µg/mL reduced endothelial tube formation and cell adhesion in co-cultured HUVECs, and decreased expression of angiogenic genes in SAECs. Similarly, exposure of differentiated SAECs to PM2.5 surrogates reduced cell reproductive ability, adhesion and tube formation of neighboring HUVECs. This indicates epithelial exposure to CB and urban PM2.5 surrogates both compromised the angiogenic ability of endothelial cells through bystander effects, thereby potentially perturbing the ventilation-perfusion ratio and affecting lung function.


Assuntos
Poluentes Atmosféricos/toxicidade , Material Particulado/toxicidade , Testes de Toxicidade , Técnicas de Cocultura , Células Epiteliais , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Pulmão/metabolismo , Neovascularização Patológica , Fuligem
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