Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.664
Filtrar
1.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199759

RESUMO

The TWIK-related spinal cord potassium channel (TRESK) is encoded by KCNK18, and variants in this gene have previously been associated with susceptibility to familial migraine with aura (MIM #613656). A single amino acid substitution in the same protein, p.Trp101Arg, has also been associated with intellectual disability (ID), opening the possibility that variants in this gene might be involved in different disorders. Here, we report the identification of KCNK18 biallelic missense variants (p.Tyr163Asp and p.Ser252Leu) in a family characterized by three siblings affected by mild-to-moderate ID, autism spectrum disorder (ASD) and other neurodevelopment-related features. Functional characterization of the variants alone or in combination showed impaired channel activity. Interestingly, Ser252 is an important regulatory site of TRESK, suggesting that alteration of this residue could lead to additive downstream effects. The functional relevance of these mutations and the observed co-segregation in all the affected members of the family expand the clinical variability associated with altered TRESK function and provide further insight into the relationship between altered function of this ion channel and human disease.


Assuntos
Alelos , Deficiência Intelectual/genética , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Canais de Potássio/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Calcineurina/metabolismo , Feminino , Genoma Humano , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ionomicina/farmacologia , Masculino , Linhagem , Canais de Potássio/química , Irmãos , Xenopus laevis/metabolismo , Adulto Jovem
2.
Nat Commun ; 12(1): 3727, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140517

RESUMO

Clonal expansion of HIV-infected cells contributes to the long-term persistence of the HIV reservoir in ART-suppressed individuals. However, the contribution from cell clones that harbor inducible proviruses to plasma viremia is poorly understood. Here, we describe a single-cell approach to simultaneously sequence the TCR, integration sites and proviral genomes from translation-competent reservoir cells, called STIP-Seq. By applying this approach to blood samples from eight participants, we show that the translation-competent reservoir mainly consists of proviruses with short deletions at the 5'-end of the genome, often involving the major splice donor site. TCR and integration site sequencing reveal that cell clones with predicted pathogen-specificity can harbor inducible proviruses integrated into cancer-related genes. Furthermore, we find several matches between proviruses retrieved with STIP-Seq and plasma viruses obtained during ART and upon treatment interruption, suggesting that STIP-Seq can capture clones that are responsible for low-level viremia or viral rebound.


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Provírus/genética , Análise de Célula Única/métodos , Viremia/virologia , Linfócitos T CD4-Positivos/virologia , DNA Viral/sangue , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Ionomicina/farmacologia , Masculino , Pessoa de Meia-Idade , Filogenia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Deleção de Sequência , Carga Viral/genética
3.
J Proteome Res ; 20(6): 3150-3164, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34008986

RESUMO

Citrullination is an important post-translational modification implicated in many diseases including rheumatoid arthritis (RA), Alzheimer's disease, and cancer. Neutrophil and mast cells have different expression profiles for protein-arginine deiminases (PADs), and ionomycin-induced activation makes them an ideal cellular model to study proteins susceptible to citrullination. We performed high-resolution mass spectrometry and stringent data filtration to identify citrullination sites in neutrophil and mast cells treated with and without ionomycin. We identified a total of 833 validated citrullination sites on 395 proteins. Several of these citrullinated proteins are important components of pathways involved in innate immune responses. Using this benchmark primary sequence data set, we developed machine learning models to predict citrullination in neutrophil and mast cell proteins. We show that our models predict citrullination likelihood with 0.735 and 0.766 AUCs (area under the receiver operating characteristic curves), respectively, on independent validation sets. In summary, this study provides the largest number of validated citrullination sites in neutrophil and mast cell proteins. The use of our novel motif analysis approach to predict citrullination sites will facilitate the discovery of novel protein substrates of protein-arginine deiminases (PADs), which may be key to understanding immunopathologies of various diseases.


Assuntos
Citrulinação , Mastócitos , Citrulina/metabolismo , Ionomicina/farmacologia , Aprendizado de Máquina , Espectrometria de Massas , Mastócitos/metabolismo , Neutrófilos/metabolismo , Desiminases de Arginina em Proteínas/genética
4.
Methods Mol Biol ; 2285: 111-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928547

RESUMO

An important hallmark for the characterisation of Th cells is their capacity for cytokine expression. In this chapter, we describe how Th cells can be restimulated polyclonally to reveal their cytokine-producing potential that can then be analysed by intracellular staining and flow cytometry.


Assuntos
Citocinas/metabolismo , Citometria de Fluxo , Análise de Célula Única , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Células Cultivadas , Humanos , Ionomicina/farmacologia , Projetos de Pesquisa , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Fluxo de Trabalho
5.
Int J Mol Sci ; 22(4)2021 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-33572290

RESUMO

Tetraspanin CD9 is widely expressed on various cell types, such as cancer cells and mesenchymal stem cells (MSCs), and/or cell-released exosomes. It has been reported that exosomal CD9 plays an important role in intercellular communications involved in cancer cell migration and metastasis. However, reports on the effect of the CD9 of MSCs or MSC-derived exosomes on cancer cell migration are still lacking. In this study, using a transwell migration assay, we found that both dextran-coated iron oxide nanoparticles (dex-IO NPs) and ionomycin stimulated exosomal CD9 expression in human MSCs (hMSCs); however, hMSCs could not deliver them to melanoma cells to affect cell migration. Interestingly, a reduced migration of melanoma cell line was observed when the ionomycin-incubated hMSC-conditioned media but not dex-IO NP-labeled hMSC-conditioned media were in the bottom chamber. In addition, we found that dex-IO NPs decreased cellular CD9 expression in hMSCs but ionomycin increased this. Simultaneously, we found that ionomycin suppressed the expression and secretion of the chemokine CCL21 in hMSCs. The silencing of CD9 demonstrated an inhibitory role of cellular CD9 in CCL21 expression in hMSCs, suggesting that ionomycin could upregulate cellular CD9 to decrease CCL21 expression and secretion of hMSCs, which would reduce the migration of B16F10, A549 and U87MG cancer cell lines due to chemoattraction reduction of CCL21. The present study not only highlights the important role of bone marrow-derived hMSCs' CD9-mediated CCL21 regulation in cancer bone metastasis but also suggests a new distinct pharmaceutical strategy for prevention or/and therapy of cancer metastasis.


Assuntos
Neoplasias Ósseas/secundário , Movimento Celular/fisiologia , Quimiocina CCL21/metabolismo , Células-Tronco Mesenquimais/metabolismo , Tetraspanina 29/metabolismo , Animais , Medula Óssea/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiocina CCL21/genética , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Exossomos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Ionomicina/farmacologia , Células-Tronco Mesenquimais/citologia , Camundongos , Comunicação Parácrina/efeitos dos fármacos , Cultura Primária de Células , Tetraspanina 29/genética , Regulação para Cima/efeitos dos fármacos
6.
BMC Vet Res ; 17(1): 44, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33482811

RESUMO

BACKGROUND: Murine is the most abundantly used as laboratory animal models. There has been a tremendous amount of research including; their evolution, growth, physiology, disease modeling as well as genomic mapping. Rats and mice are the most widely used among them. Although both rats and mice fall under the same category still both are different a lot too. As regarding in vitro maturation and development mouse studies are well established as compared to rats which still lies in the early phase of development. So, we tried to figure out rat oocytes in vitro maturation and their developmental potential by performing 3 experiments i.e. superovulation, in vitro Maturation as simple culture (COC's only), and COC's & cumulus cells co-culture, which later further developed using parthenogenetic activation after IVM. Female Sprague Dawley rat 3-4 week used for these studies, we hyper-stimulated their ovaries using PMSG and hCG 150 IU/kg each. After that, we collected ovaries via dissection and retrieved oocytes. We matured them in TCM 199 supplemented with FSH, Estrogen, EGF, and Pyruvate. After maturation, we activated them using two types of activators i.e. Ethanol 7%, Ionomycin. After that, we saw and compared their developmental potential in vitro. RESULTS: Oocytes matured in COC's and Cumulus cell monolayer co-culture (59% ± 4*) showed significantly more even growth and extrusion of the first polar body as compared to the COC's only culture (53.8 ± 7%*). While oocytes activated using Ionomycin showed more promising development until 8 cells/blastocyst level compared to ethanol 7%. CONCLUSION: we concluded that COC's and cumulus monolayer co-culture is better than COC's only culture. Cumulus monolayer provides extra aid in the absorption of nutrients and supplements thus providing a better environment for oocytes growth. Also, we concluded that matured oocytes showed more developmental capacity after activation via ionomycin compared to ethanol.


Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Animais , Técnicas de Cocultura/métodos , Técnicas de Cocultura/veterinária , Meios de Cultura , Células do Cúmulo/citologia , Células do Cúmulo/fisiologia , Etanol/farmacologia , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Ionomicina/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Partenogênese , Ratos Sprague-Dawley
7.
Methods Mol Biol ; 2270: 61-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33479893

RESUMO

IL-10 is the best known and most studied anti-inflammatory cytokine and, in the last 20 years, it has acquired even greater fame as it has been associated with the regulatory phenotype of B cells. Indeed, although great efforts have been made to find a unique marker, to date IL-10 remains the main way to follow both murine and human regulatory B cells, hence the need of precise and reproducible methods to identify and purify IL-10-producing B cells for both functional and molecular downstream assays. In this chapter, we present our protocols to isolate these cells from the murine spleen and peritoneum and from human peripheral blood. Since the production of IL-10 by B cells is not only a weapon to counteract the adverse effect of pro-inflammatory cytokines but also a response to cellular activation, we focused on those B cells that are prone to IL-10 production and detectable following a short-term stimulation with phorbol-12-myristate-13-acetate, ionomycin, and lipopolysaccharide (murine system) or CpG (human system).


Assuntos
Subpopulações de Linfócitos B/citologia , Linfócitos B Reguladores/citologia , Separação Celular/métodos , Animais , Subpopulações de Linfócitos B/imunologia , Citocinas/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Humanos , Interleucina-10/metabolismo , Ionomicina/farmacologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/imunologia , Camundongos , Ésteres de Forbol/farmacologia , Baço/citologia , Acetato de Tetradecanoilforbol/farmacologia
8.
Rheumatology (Oxford) ; 60(4): 1687-1699, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33026085

RESUMO

OBJECTIVES: Patients with APS are at increased risk of thromboembolism. Neutrophils have been shown to play a role in inducing thrombosis. We aimed to investigate differences in neutrophil subpopulations, their potential of activation and neutrophil extracellular trap (NET) formation comparing high and low-density neutrophils (HDNs/LDNs) as well as subpopulations in patients with APS and controls to gain deeper insight into their potential role in thrombotic manifestations in patients with APS. METHODS: HDNs and LDNs of 20 patients with APS and 20 healthy donors were isolated by density gradient centrifugation and stimulated. Neutrophil subpopulations, their activation and NET release were assessed by flow cytometry. RESULTS: LDNs of both groups showed higher baseline activation, lower response to stimulation (regulation of activation markers CD11b/CD66b), but higher NET formation compared with HDNs. In patients with APS, the absolute number of LDNs was higher compared with controls. HDNs of APS patients showed higher spontaneous activation [%CD11b high: median (interquartile range): 2.78% (0.58-10.24) vs 0.56% (0.19-1.37)] and response to stimulation with ionomycin compared with HDNs of healthy donors [%CD11b high: 98.20 (61.08-99.13) vs 35.50% (13.50-93.85)], whereas no difference was found in LDNs. NET formation was increased in patients' HDNs upon stimulation. CONCLUSION: HDNs and LDNs act differently, unstimulated and upon various stimulations in both healthy controls and APS patients. Differences in HDNs and LDNs between patients with APS and healthy controls indicate that neutrophils may enhance the risk of thrombosis in these patients and could thus be a target for prevention of thrombosis in APS.


Assuntos
Síndrome Antifosfolipídica/metabolismo , Armadilhas Extracelulares/metabolismo , Ativação de Neutrófilo , Neutrófilos/metabolismo , Adulto , Anticorpos/sangue , Antígenos CD/metabolismo , Antígeno CD11b/metabolismo , Estudos de Casos e Controles , Moléculas de Adesão Celular/metabolismo , Estudos de Coortes , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Ionomicina/farmacologia , Inibidor de Coagulação do Lúpus/sangue , Masculino , Pessoa de Meia-Idade , beta 2-Glicoproteína I/imunologia
9.
Biochimie ; 181: 169-175, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33333171

RESUMO

We investigated whether docosahexaenoic acid (DHA), a dietary n-3 fatty acid, modulates calcium (Ca2+) signaling and cell cycle progression in human Jurkat T-cells. Our study demonstrates that DHA inhibited Jurkat T-cell cycle progression by blocking their passage from S phase to G2/M phase. In addition, DHA decreased the plasma membrane expression of TRPC3 and TRPC6 calcium channels during T-cell proliferation. Interestingly, this fatty acid increased plasma membrane expression of TRPC6 after 24 h of mitogenic stimulation by phorbol-13-myristate-12-acetate (PMA) and ionomycin. These variations in the membrane expression of TRPC3 and TRPC6 channels were not directly correlated with the mRNA expression, indicating that it was a post-translational phenomenon. DHA increased free intracellular calcium concentrations, [Ca2+]i, via opening TRPC3 and TRPC6 channels. We conclude that the anti-proliferative effect of DHA might involve the modulation of TRPC3 and TRPC6 channels in human T-cells.


Assuntos
Membrana Celular/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Linfócitos T/metabolismo , Canais de Cátion TRPC/biossíntese , Canal de Cátion TRPC6/biossíntese , Humanos , Ionomicina/farmacologia , Células Jurkat , Acetato de Tetradecanoilforbol/farmacologia
10.
Int Immunopharmacol ; 88: 106992, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33182021

RESUMO

OBJECTIVE: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocytic infiltration of the exocrine glands. Recent, studies have shown that the long noncoding RNA (lncRNA) NEAT1 plays a crucial role in regulating the immune response. However, studies on the lncRNA NEAT1 in pSS are limited. Exploring the role of the lncRNA NEAT1 in the pathogenesis of pSS was the purpose of this study. METHODS: The expression of NEAT1 in peripheral blood mononuclear cells (PBMCs) of patients with pSS and healthy controls (HCs) was analyzed by real-time polymerase chain reaction (RT-PCR). Antisense oligonucleotides (ASOs) and siRNA or immune stimulation with PMA/ionomycin were used to perform loss-and-gain-of-function experiments. RT-PCR, enzyme-linked immunosorbent assay (ELISA), and Western blot were performed to detect the RNA and protein levels of specific genes induced by PMA/ionomycin stimulation. Microarray analysis was used to generate an overview of the genes that might be regulated by NEAT1. RESULTS: Compared with that in HC patient cells, the expression of NEAT1 in pSS patients was mainly increased in peripheral T cells, including CD4+ and CD8+ T cells. Additionally, the expression of NEAT1 in CD4+ T cells of patients with pSS was positively correlated with the course of disease. NEAT1 expression in Jurkat cells was induced by PMA/ionomycin stimulation upon activation of the TCR-p38 pathway. Upregulation of NEAT1 expression also increased the expression of CXCL8 and TNF-α. Knocking down NEAT1 expression with an ASO suppressed the expression of CXCL8 and TNF-α in PMA/ionomycin-stimulated Jurkat cells. Then, we found that NEAT1 regulated the activation of MAPK pathway to regulate NEAT1-induced factors, selectively activating the expression of p-p38 and p-ERK1/2. Furthermore, we also detected the expression profile of Jurkat cells stimulated by PMA/ionomycin when NEAT1 was silenced or not, in order to produce an overview of NEAT1-regulated genes. CONCLUSION: These results provide a new understanding of the mechanisms of pSS and reveal that NEAT1 is a positive regulator of pSS, which is of substantial significance to its pathogenesis. Thus, NEAT1 provides a potential therapeutic target for pSS.


Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , RNA Longo não Codificante/metabolismo , Síndrome de Sjogren/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ionomicina/farmacologia , Células Jurkat , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , RNA Longo não Codificante/genética , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Síndrome de Sjogren/metabolismo , Regulação para Cima
11.
Retrovirology ; 17(1): 30, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32912211

RESUMO

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) infects primarily CD4+ T-lymphocytes and evoques severe diseases, predominantly Adult T-Cell Leukemia/ Lymphoma (ATL/L) and HTLV-1-associated Myelopathy/ Tropical Spastic Paraparesis (HAM/TSP). The viral transactivator of the pX region (Tax) is important for initiating malignant transformation, and deregulation of the major signaling pathway nuclear factor of kappa B (NF-κB) by Tax represents a hallmark of HTLV-1 driven cancer. RESULTS: Here we found that Tax mutants which are defective in NF-κB signaling showed diminished protein expression levels compared to Tax wildtype in T-cells, whereas Tax transcript levels were comparable. Strikingly, constant activation of NF-κB signaling by the constitutive active mutant of inhibitor of kappa B kinase (IKK2, IKK-ß), IKK2-EE, rescued protein expression of the NF-κB defective Tax mutants M22 and K1-10R and even increased protein levels of Tax wildtype in various T-cell lines while Tax transcript levels were only slightly affected. Using several Tax expression constructs, an increase of Tax protein occurred independent of Tax transcripts and independent of the promoter used. Further, Tax and M22 protein expression were strongly enhanced by 12-O-Tetradecanoylphorbol-13-Acetate [TPA; Phorbol 12-myristate 13-acetate (PMA)]/ ionomycin, inducers of NF-κB and cytokine signaling, but not by tumor necrosis factor alpha (TNF-α). On the other hand, co-expression of Tax with a dominant negative inhibitor of κB, IκBα-DN, or specific inhibition of IKK2 by the compound ACHP, led to a vast decrease in Tax protein levels to some extent independent of Tax transcripts in transiently transfected and Tax-transformed T-cells. Cycloheximide chase experiments revealed that co-expression of IKK2-EE prolongs the half-life of M22, and constant repression of NF-κB signaling by IκBα-DN strongly reduces protein stability of Tax wildtype suggesting that NF-κB activity is required for Tax protein stability. Finally, protein expression of Tax and M22 could be recovered by NH4Cl and PYR-41, inhibitors of the lysosome and the ubiquitin-activating enzyme E1, respectively. CONCLUSIONS: Together, these findings suggest that Tax's capability to induce NF-κB is critical for protein expression and stabilization of Tax itself. Overall, identification of this novel positive feedback loop between Tax and NF-κB in T-cells improves our understanding of Tax-driven transformation.


Assuntos
Retroalimentação Fisiológica , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Subunidade p50 de NF-kappa B/metabolismo , Regulação da Expressão Gênica , Produtos do Gene tax/genética , Humanos , Ionomicina/farmacologia , Células Jurkat , Mutação , Subunidade p50 de NF-kappa B/genética , Estabilidade Proteica , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia
12.
J Virol ; 94(21)2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32817219

RESUMO

Adeno-associated viruses (AAVs) are dependoparvoviruses that have proven useful for therapeutic gene transfer; however, our understanding of host factors that influence AAV trafficking and transduction is still evolving. Here, we investigated the role of cellular calcium in the AAV infectious pathway. First, we demonstrated a critical role for the host Golgi compartment-resident ATP-powered calcium pump (secretory pathway calcium ATPase 1 [SPCA1]) encoded by the ATP2C1 gene in AAV infection. CRISPR-based knockout (KO) of ATP2C1 decreases transduction by different AAV serotypes. ATP2C1 KO does not appear to inhibit AAV binding, cellular uptake, or nuclear entry; however, capsids within ATP2C1 KO cells demonstrate dispersed and punctate trafficking distinct from the perinuclear, trans-Golgi pattern observed in normal cells. In addition, we observed a defect in the ability of AAV capsids to undergo conformational changes and support efficient vector genome transcription in ATP2C1 KO cells. The calcium chelator BAPTA-AM, which reduces cytosolic calcium, rescues the defective ATP2C1 KO phenotype and AAV transduction in vitro Conversely, the calcium ionophore ionomycin, which disrupts calcium gradients, blocks AAV transduction. Further, we demonstrated that modulating calcium in the murine brain using BAPTA-AM augments AAV gene expression in vivo Taking these data together, we postulate that the maintenance of an intracellular calcium gradient by the calcium ATPase and processing within the Golgi compartment are essential for priming the capsid to support efficient AAV genome transcription.IMPORTANCE Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigated the role of ATP2C1, which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency.


Assuntos
ATPases Transportadoras de Cálcio/genética , Cálcio/metabolismo , Dependovirus/genética , Vetores Genéticos/metabolismo , Complexo de Golgi/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Sistemas CRISPR-Cas , ATPases Transportadoras de Cálcio/deficiência , Linhagem Celular Tumoral , Quelantes/farmacologia , Dependovirus/efeitos dos fármacos , Dependovirus/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Deleção de Genes , Vetores Genéticos/química , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/virologia , Células HEK293 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Injeções Intraventriculares , Ionomicina/farmacologia , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Técnicas Estereotáxicas , Transdução Genética , Vesiculovirus/genética , Vesiculovirus/metabolismo
13.
Sci Rep ; 10(1): 11694, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678135

RESUMO

Neutrophils are pivotal players in immune defence which includes a process of release of histones and DNA as neutrophil extracellular traps (NETs). Histones, while toxic to invading pathogens, also kill host cells, including neutrophils. Bacteria have evolved mechanisms to escape neutrophils, including the secretion of leucocidins (e.g. ionomycin). Live cell video microscopy showed how fibrinogen and fibrin influence NETosis and neutrophil responses to extracellular histones. Histones were rapidly lethal to neutrophils after binding to cells, but formation of fibrinogen/fibrin-histone aggregates prevented cell death. Histone cytotoxicity was also reduced by citrullination by peptidyl arginine deiminase 4, or digestion by serine proteases. Ionomycin and phorbol 12-myristate 13 acetate (PMA) are used to trigger NETosis. Fibrinogen was responsible for a second distinct mechanism of neutrophil protection after treatment with ionomycin. Fibrinogen clustered on the surface of ionomycin-stimulated neutrophils to delay NETosis; and blocking the ß integrin receptor, αMß2, abolished fibrinogen protection. Fibrinogen did not bind to or protect neutrophils stimulated with PMA. Fibrinogen is an acute phase protein that will protect exposed cells from damaging circulating histones or leucocidins; but fibrinogen depletion/consumption, as in trauma or sepsis will reduce protection. It is necessary to consider the role of fibrinogen in NETosis.


Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Produtos de Degradação da Fibrina e do Fibrinogênio/farmacologia , Histonas/farmacologia , Ionomicina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Substâncias Protetoras/farmacologia , Doadores de Sangue , Morte Celular/efeitos dos fármacos , Células Cultivadas , Citrulinação , DNA/metabolismo , Armadilhas Extracelulares/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Histonas/metabolismo , Humanos , Antígeno de Macrófago 1/metabolismo , Agregados Proteicos , Acetato de Tetradecanoilforbol/farmacologia
14.
Sci Rep ; 10(1): 9014, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488068

RESUMO

The uterus plays an important and unique role during pregnancy and is a dynamic organ subjected to mechanical stimuli. It has been reported that infertility occurs when the peristalsis is prevented, although its mechanisms remain unknown. In this study, we found that mechanical strain mimicking the peristaltic motion of the uterine smooth muscle layer enabled the endometrial stromal cells to acquire contractility. In order to mimic the peristalsis induced by uterine smooth muscle cells, cyclic tensile stretch was applied to human endometrial stromal cells. The results showed that the strained cells exerted greater contractility in three-dimensional collagen gels in the presence of oxytocin, due to up-regulated alpha-smooth muscle actin expression via the cAMP signaling pathway. These in vitro findings underscore the plasticity of the endometrial stromal cell phenotype and suggest the possibility of acquired contractility by these cells in vivo and its potential contribution to uterine contractile activity. This phenomenon may be a typical example of how a tissue passively acquires new contractile functions under mechanical stimulation from a neighboring tissue, enabling it to support the adjacent tissue's functions.


Assuntos
Endométrio/citologia , Miócitos de Músculo Liso/citologia , Células Estromais/fisiologia , Resistência à Tração , Actinas/antagonistas & inibidores , Actinas/metabolismo , Adulto , Células Cultivadas , Colágeno Tipo I/metabolismo , AMP Cíclico/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Ionomicina/farmacologia , Isoquinolinas/farmacologia , Pessoa de Meia-Idade , Músculo Liso , Ocitocina/farmacologia , Peristaltismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Sulfonamidas/farmacologia , Regulação para Cima
15.
Int J Mol Sci ; 21(11)2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32517157

RESUMO

Anoctamins such as TMEM16A and TMEM16B are Ca2+-dependent Cl- channels activated through purinergic receptor signaling. TMEM16A (ANO1), TMEM16B (ANO2) and TMEM16F (ANO6) are predominantly expressed at the plasma membrane and are therefore well accessible for functional studies. While TMEM16A and TMEM16B form halide-selective ion channels, TMEM16F and probably TMEM16E operate as phospholipid scramblases and nonselective ion channels. Other TMEM16 paralogs are expressed mainly in intracellular compartments and are therefore difficult to study at the functional level. Here, we report that TMEM16E (ANO5), -H (ANO8), -J (ANO9) and K (ANO10) are targeted to the plasma membrane when fused to a C-terminal CAAX (cysteine, two aliphatic amino acids plus methionin, serine, alanin, cystein or glutamin) motif. These paralogs produce Ca2+-dependent ion channels. Surprisingly, expression of the TMEM16 paralogs in the plasma membrane did not produce additional scramblase activity. In contrast, endogenous scrambling induced by stimulation of purinergic P2X7 receptors was attenuated, in parallel with reduced plasma membrane blebbing. This could suggest that intracellular TMEM16 paralogs operate differently when compared to plasma membrane-localized TMEM16F, and may even stabilize intracellular membranes. Alternatively, CAAX tagging, which leads to expression in non-raft compartments of the plasma membrane, may antagonize phosphatidylserine exposure by endogenous raft-located TMEM16F. CAAX-containing constructs may be useful to further investigate the molecular properties of intracellular TMEM16 proteins.


Assuntos
Anoctaminas/metabolismo , Membrana Celular/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Imunofluorescência , Expressão Gênica , Espaço Intracelular/metabolismo , Ionomicina/farmacologia , Família Multigênica , Fosfolipídeos/metabolismo , Ratos , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais/efeitos dos fármacos
16.
Cells ; 9(6)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32521784

RESUMO

Cytokines are the major immune regulators secreted from activated CD4+ T lymphocytes that activate adaptive immunity to eradicate nonself cells, including pathogens, tumors, and allografts. The regulation of glycogen synthase kinase (GSK)-3ß, a serine/threonine kinase, controls cytokine production by regulating transcription factors. The artificial in vitro activation of CD4+ T lymphocytes by a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin, the so-called T/I model, led to an inducible production of cytokines, such as interferon-γ, tumor necrosis factor-α, and interleukin-2. As demonstrated by the approaches of pharmacological targeting and genetic knockdown of GSK-3ß, T/I treatment effectively caused GSK-3ß activation followed by GSK-3ß-regulated cytokine production. In contrast, pharmacological inhibition of the proline-rich tyrosine kinase 2 and calcineurin signaling pathways blocked cytokine production, probably by deactivating GSK-3ß. The blockade of GSK-3ß led to the inhibition of the nuclear translocation of T-bet, a vital transcription factor of T lymphocyte cytokines. In a mouse model, treatment with the GSK-3ß inhibitor 6-bromoindirubin-3'-oxime significantly inhibited T/I-induced mortality and serum cytokine levels. In summary, targeting GSK-3ß effectively inhibits CD4+ T lymphocyte activation and cytokine production.


Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/biossíntese , Glicogênio Sintase Quinase 3 beta/metabolismo , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Calcineurina/metabolismo , Linhagem da Célula/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Quinase 2 de Adesão Focal/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Humanos , Masculino , Camundongos Endogâmicos C57BL , Transporte Proteico , Transdução de Sinais/efeitos dos fármacos , Proteínas com Domínio T/metabolismo
17.
Cell Physiol Biochem ; 54(4): 605-614, 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32543797

RESUMO

BACKGROUND/AIMS: Suicidal erythrocyte death (eryptosis) is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface following a Ca2+ entry in the cell. Eryptosis is stimulated by increased cytosolic Ca2+ ([Ca2+]i), oxidative stress, energy depletion, or high osmotic shock. Eryptosis signaling includes p38 mitogen-activated protein kinase (MAPK), caspases, casein kinase 1 (CK1), janus kinase 3 (JAK3), and protein kinase C (PKC). Dog and human erythrocytes have different characteristics, for example, dog erythrocytes lack Na+/K+- ATPase activity. Whether eryptosis occurs in dog erythrocytes in an analogous way as that in humans remains unclear. Eryptosis in dogs has not been investigated. This study aimed to explore which stimulator and signaling molecules are involved in eryptosis in healthy dog erythrocytes. METHODS: Erythrocytes were isolated from 10 dogs, and eryptosis was stimulated by oxidative stress with tert-butyl hydroperoxide (tBOOH), high osmotic shock with excessive sucrose condition, energy depletion with minus glucose condition, and high [Ca2+]i with ionomycin. Phosphatidylserine exposure was estimated using annexin V binding. Erythrocyte volume and [Ca2+]i were measured by forward scatter and Fluo3-fluorescence, respectively. In addition, the role of certain mediators was assessed using the following inhibitors to determine the detailed mechanisms of eryptosis in dog erythrocytes: p38MAPK, caspase family, CK1, JAK3, and PKC inhibitors. RESULTS: All eryptosis-inducing factors resulted in phosphatidylserine exposures, except for ionomycin. In addition, the erythrocyte volume increased with ionomycin and tBOOH but decreased with excessive sucrose and minus glucose condition. All treatments increased [Ca2+]i. Furthermore, WH1-P154 and chelerythrine significantly blunted the increase of annexin V binding erythrocytes following the tBOOH treatment. CONCLUSION: Eryptosis in dogs is triggered by oxidative stress, hyperosmotic shock, and energy depletion. It is suggested that JAK3 and PKC play an important role in eryptosis following an oxidative stress in dog erythrocytes.


Assuntos
Cálcio/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Fosfatidilserinas/metabolismo , terc-Butil Hidroperóxido/farmacologia , Animais , Anexina A5/metabolismo , Benzofenantridinas/farmacologia , Caseína Quinase I/antagonistas & inibidores , Inibidores de Caspase , Caspases/metabolismo , Tamanho Celular/efeitos dos fármacos , Cães , Eriptose , Glucose/metabolismo , Ionomicina/farmacologia , Janus Quinase 3/antagonistas & inibidores , Pressão Osmótica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sacarose/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
18.
Cells ; 9(5)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32438725

RESUMO

The mechanisms underlying the allergy-protective effects of raw cow's milk are poorly understood. The current focus is mainly on the modulation of T cell responses. In the present study, we investigated whether raw cow's milk can also directly inhibit mast cells, the key effector cells in IgE-mediated allergic responses. Primary murine bone marrow-derived mast cells (BMMC) and peritoneal mast cells (PMC), were incubated with raw milk, heated raw milk, or shop milk, prior to IgE-mediated activation. The effects on mast cell activation and underlying signaling events were assessed. Raw milk was furthermore fractionated based on molecular size and obtained fractions were tested for their capacity to reduce IgE-mediated mast cell activation. Coincubation of BMMC and PMC with raw milk prior to activation reduced ß-hexosaminidase release and IL-6 and IL-13 production, while heated raw milk or shop milk had no effect. The reduced mast cell activation coincided with a reduced intracellular calcium influx. In addition, SYK and ERK phosphorylation levels, both downstream signaling events of the FcεRI, were lower in raw milk-treated BMMC compared to control BMMC, although differences did not reach full significance. Raw milk-treated BMMC furthermore retained membrane-bound IgE expression after allergen stimulation. Raw milk fractionation showed that the heat-sensitive raw milk components responsible for the reduced mast cell activation are likely to have a molecular weight of > 37 kDa. The present study demonstrates that raw cow's milk can also directly affect mast cell activation. These results extend the current knowledge on mechanisms via which raw cow's milk prevents allergic diseases, which is crucial for the development of new, microbiologically safe, nutritional strategies to reduce allergic diseases.


Assuntos
Hipersensibilidade/imunologia , Leite/efeitos adversos , Alérgenos/imunologia , Animais , Cálcio/metabolismo , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Imunoglobulina E/metabolismo , Ionomicina/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Receptores de IgE/metabolismo , Quinase Syk/metabolismo
19.
Int J Mol Sci ; 21(11)2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32471032

RESUMO

Tumor-infiltrating CD8+ T cells (TIL) are of the utmost importance in anti-tumor immunity. CD103 defines tumor-resident memory T cells (TRM cells) associated with improved survival and response to immune checkpoint blockade (ICB) across human tumors. Co-expression of CD39 and CD103 marks tumor-specific TRM with enhanced cytolytic potential, suggesting that CD39+CD103+ TRM could be a suitable biomarker for immunotherapy. However, little is known about the transcriptional activity of TRM cells in situ. We analyzed CD39+CD103+ TRM cells sorted from human high-grade endometrial cancers (n = 3) using mRNA sequencing. Cells remained untreated or were incubated with PMA/ionomycin (activation), actinomycin D (a platinum-like chemotherapeutic that inhibits transcription), or a combination of the two. Resting CD39+CD103+ TRM cells were transcriptionally active and expressed a characteristic TRM signature. Activated CD39+CD103+ TRM cells differentially expressed PLEK, TWNK, and FOS, and cytokine genes IFNG, TNF, IL2, CSF2 (GM-CSF), and IL21. Findings were confirmed using qPCR and cytokine production was validated by flow cytometry of cytotoxic TIL. We studied transcript stability and found that PMA-responsive genes and mitochondrial genes were particularly stable. In conclusion, CD39+CD103+ TRM cells are transcriptionally active TRM cells with a polyfunctional, reactivation-responsive repertoire. Secondly, we hypothesize that differential regulation of transcript stability potentiates rapid responses upon TRM reactivation in tumors.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias do Endométrio/imunologia , Neoplasias do Endométrio/patologia , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Dactinomicina/farmacologia , Neoplasias do Endométrio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genótipo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Interleucinas/metabolismo , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Gradação de Tumores , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Genética/efeitos dos fármacos
20.
Arthritis Rheumatol ; 72(8): 1303-1313, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32243724

RESUMO

OBJECTIVE: Interleukin-17A (IL-17A) and tumor necrosis factor (TNF) contribute to the pathogenesis of psoriatic arthritis (PsA). However, their functional relationship in PsA synovitis has not been fully elucidated. Additionally, although CD8+ T cells in PsA have been recognized via flow cytometry as a source of IL-17A production, it is not clear whether CD8+ T cells secrete IL-17A under more physiologically relevant conditions in the context from PsA synovitis. This study was undertaken to clarify the roles of IL-17A and TNF in the synovial fluid (SF) from patients with PsA and investigate the impact of CD8+ T cells on IL-17A production. METHODS: IL-17A+ T cells were identified by flow cytometry in SF samples from 20 patients with active PsA, blood samples from 22 treatment-naive patients with PsA, and blood samples from 22 healthy donors. IL-17A+ T cells were sorted from 12 PsA SF samples and stimulated using anti-CD3/anti-CD28 or phorbol myristate acetate (PMA) and ionomycin ex vivo, alone (n = 3) or together with autologous monocytes (n = 3) or PsA fibroblast-like synoviocytes (FLS) (n = 5-6). To evaluate the differential allogeneic effects of neutralizing IL-17A and TNF, SF CD4+ T cells and PsA FLS cocultures were also used (n = 5-6). RESULTS: Flow cytometry analyses of SF samples from patients with PsA showed IL-17A positivity for CD4+ and CD8+ T cells (IL-17A, median 0.71% [interquartile range 0.35-1.50%] in CD4+ cells; median 0.44% [interquartile range 0.17-1.86%] in CD8+ T cells). However, only CD4+ T cells secreted IL-17A after anti-CD3/anti-CD28 activation, when cultured alone and in cocultures with PsA monocytes or PsA FLS (each P < 0.05). Remarkably, CD8+ T cells only secreted IL-17A after 4- or 72-hour stimulation with PMA/ionomycin. Anti-IL-17A and anti-TNF treatments both inhibited PsA synovitis ex vivo. Neutralizing IL-17A strongly inhibited IL-6 (P < 0.05) and IL-1ß (P < 0.01), while anti-TNF treatment was more potent in reducing matrix metalloproteinase 3 (MMP-3) (P < 0.05) and MMP-13. CONCLUSION: CD8+ T cells, in contrast to CD4+ T cells, in SF specimens obtained from PsA patients did not secrete IL-17A following T cell receptor activation. Overlapping, but distinct, effects at the level of inflammatory cytokines and MMPs were found after neutralizing IL-17A or TNF ex vivo in a human model of PsA synovitis.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interleucina-17/biossíntese , Receptores de Antígenos de Linfócitos T/administração & dosagem , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Artrite Psoriásica/tratamento farmacológico , Artrite Psoriásica/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cultura de Células , Feminino , Citometria de Fluxo , Humanos , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Receptores de Antígenos de Linfócitos T/imunologia , Líquido Sinovial , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/imunologia , Sinovite/tratamento farmacológico , Sinovite/imunologia , Acetato de Tetradecanoilforbol/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...