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- ItemAcesso aberto (Open Access)SARS-CoV-2 infection and replication kinetics in different human cell types: The role of autophagy, cellular metabolism and ACE2 expression(Elsevier, 2022-09-06) Bartolomeo, Cynthia Silva [UNIFESP]; Lemes, Robertha Mariana Rodrigues [UNIFESP]; Morais, Rafael Leite Tavares de [UNIFESP]; Pereira, Gabriela Cruz [UNIFESP]; Nunes, Tamires Alves [UNIFESP]; Costa, Angelica Jardim [UNIFESP]; Maciel, Rui Monteiro de Barros [UNIFESP]; Braconi, Carla Torres [UNIFESP]; Maricato, Juliana Terzi [UNIFESP]; Janini, Luiz Mário Ramos [UNIFESP]; Okuda, Liria Hiromi; Lee, Kil Sun [UNIFESP]; Prado , Carla Máximo [UNIFESP]; Ureshino, Rodrigo Portes [UNIFESP]; Stilhano, Roberta Sessa; http://lattes.cnpq.br/1740478426977844; http://lattes.cnpq.br/9682597956704119; http://lattes.cnpq.br/5975254446394746; http://lattes.cnpq.br/1212794444821641; http://lattes.cnpq.br/2542981501423374; http://lattes.cnpq.br/8517445237869609; http://lattes.cnpq.br/1005025547870062; http://lattes.cnpq.br/3864261034300240; http://lattes.cnpq.br/6342740138292278; http://lattes.cnpq.br/8321096323728598; http://lattes.cnpq.br/5713863164263481; http://lattes.cnpq.br/4546671040397891; http://lattes.cnpq.br/7705881286363327; http://lattes.cnpq.br/1740478426977844; http://lattes.cnpq.br/7174742745591377; http://lattes.cnpq.br/2122125365795721; Universidade Federal de São Paulo (UNIFESP)Aims: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and inves tigated molecular mechanisms related to viral infection susceptibility and replication. Main methods: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 over expression on viral replication in pulmonary cells was also investigated. Key findings: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. Significance: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.
- ItemEmbargoAlpha-7 nicotinic receptor agonist protects mice against pulmonary emphysema induced by elastase(Springer Nature, 2024-06-01) Banzato, Rosana; Pinheiro-Menegasso, Nathalia Montouro [UNIFESP]; Santana, Fernanda Paula Roncon [UNIFESP]; Olivo, Clarice Rosa; Taguchi, Laura [UNIFESP]; Santos, Stheffany de Oliveira [UNIFESP]; Fukuzaki, Silvia; Teodoro, Walcy Paganelli Rosolia; Lopes, Fernanda Degobbi Tenorio Quirino dos Santos; Tibério, Iolanda de Fátima Lopes Calvo; Toledo-Arruda, Alessandra Choqueta de; Prado, Marco Antônio Máximo; Prado, Vania Ferreira; Prado , Carla Máximo [UNIFESP]; http://lattes.cnpq.br/9600880359624334; http://lattes.cnpq.br/8960401765088965; http://lattes.cnpq.br/0335008572194595; http://lattes.cnpq.br/0090429486050126; http://lattes.cnpq.br/4409788652533408; http://lattes.cnpq.br/1848955229237487; http://lattes.cnpq.br/0720259963845051; http://lattes.cnpq.br/2069814538238357; http://lattes.cnpq.br/7240963502716211; http://lattes.cnpq.br/8148205870336524; http://lattes.cnpq.br/2961494666434143; http://lattes.cnpq.br/8706664565242249; http://lattes.cnpq.br/3222014752923501; http://lattes.cnpq.br/1740478426977844; Universidade Federal de São Paulo (UNIFESP)Pulmonary emphysema is a primary component of chronic obstructive pulmonary disease (COPD), a life-threatening disorder characterized by lung inflammation and restricted airflow, primarily resulting from the destruction of small airways and alveolar walls. Cumulative evidence suggests that nicotinic receptors, especially the α7 subtype (α7nAChR), is required for anti-inflammatory cholinergic responses. We postulated that the stimulation of α7nAChR could offer therapeutic benefits in the context of pulmonary emphysema. To investigate this, we assessed the potential protective effects of PNU-282987, a selective α7nAChR agonist, using an experimental emphysema model. Male mice (C57BL/6) were submitted to a nasal instillation of porcine pancreatic elastase (PPE) (50 µl, 0.667 IU) to induce emphysema. Treatment with PNU-282987 (2.0 mg/kg, ip) was performed pre and post-emphysema induction by measuring anti-inflammatory effects (inflammatory cells, cytokines) as well as anti-remodeling and anti-oxidant effects. Elastase-induced emphysema led to an increase in the number of α7nAChR-positive cells in the lungs. Notably, both groups treated with PNU-282987 (prior to and following emphysema induction) exhibited a significant decrease in the number of α7nAChR-positive cells. Furthermore, both groups treated with PNU-282987 demonstrated decreased levels of macrophages, IL-6, IL-1β, collagen, and elastic fiber deposition. Additionally, both groups exhibited reduced STAT3 phosphorylation and lower levels of SOCS3. Of particular note, in the post-treated group, PNU-282987 successfully attenuated alveolar enlargement, decreased IL-17 and TNF-α levels, and reduced the recruitment of polymorphonuclear cells to the lung parenchyma. Significantly, it is worth noting that MLA, an antagonist of α7nAChR, counteracted the protective effects of PNU-282987 in relation to certain crucial inflammatory parameters. In summary, these findings unequivocally demonstrate the protective abilities of α7nAChR against elastase-induced emphysema, strongly supporting α7nAChR as a pivotal therapeutic target for ameliorating pulmonary emphysema.
- ItemAcesso aberto (Open Access)Antineoplastic drugs in environmentally relevant concentrations cause endocrine disruption and testicular dysfunction in experimental conditions(Elsevier, 2023-04-07) Medeiros, Paloma da Cunha de [UNIFESP]; Perobelli, Juliana Elaine [UNIFESP]; Nascimento, Cinthia Castro do [UNIFESP]; https://lattes.cnpq.br/9844283096155413; http://lattes.cnpq.br/2047233951021632; Universidade Federal de São Paulo (UNIFESP)5-fluorouracil (5-FU) and methotrexate (MTX) are among the most widely consumed antineoplastic drugs worldwide. These drugs are known as emerging pollutants, once after consumption are excreted by feces and/or urine in a mixture of compounds and metabolites, entering the aquatic environment due to low efficiency in drug removal by effluent treatment plants. Considering that these substances may interact with the DNA, causing metabolic and morphological changes, leading to cell death, the present study aimed to investigate the potential impact of a long-term exposure to these antineoplastic drugs in environmentally relevant concentrations, on testicular morphophysiology of rats. Male Wistar rats (70 days old) were distributed into 4 groups (n = 10 / group): control, received only vehicle; MTX, received methotrexate at 10ng/L in drinking water; 5-FU received 5-fluorouracil at 10ng/L in drinking water; and MTX+ 5FU, received the combination of MTX and 5-FU at 10ng/L each. The treatment period was from postnatal day (PND)70 to PND160, when the animals were euthanized for evaluation of testicular toxicity and changes in endocrine signaling. In these experimental conditions, both drugs acted as endocrine disruptors causing cytotoxic effects in the testes of exposed rats, altering the structural pattern of seminiferous tubules and leading to oxidative stress even at environmental concentrations.
- ItemAcesso aberto (Open Access)Acute Lung Injury in Cholinergic-Deficient Mice Supports Anti-Inflammatory Role of α7 Nicotinic Acetylcholine Receptor(Nicola Scichilone, 2021-07) Pinheiro, Nathalia Montouro [UNIFESP]; Banzato, Rosana; Tibério, Iolanda de Fátima Lopes Calvo; Prado, Marco Antônio Máximo; Prado, Vania Ferreira; Hamouda, Ayman; Prado, Carla Máximo [UNIFESP]; http://lattes.cnpq.br/8960401765088965Abstract: (1) Background: The lung cholinergic pathway is important for controlling pulmonary inflammation in acute lung injury, a condition that is characterized by a sudden onset and intense inflammation. This study investigated changes in the expression levels of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) in the lung during acute lung injury. (2) Methods: acute lung injury (ALI) was induced in wild-type and cholinergic-deficient (VAChT-KDHOM) mice using intratracheal lipopolysaccharide (LPS) instillation with or without concurrent treatment with nicotinic ligands. Bronchoalveolar lavage fluid was collected to evaluate markers of inflammation, and then the lung was removed and processed for isolation of membrane fraction and determination of acetylcholine receptors level using radioligand binding assays. (3) Results: LPS-induced increase in lung inflammatory markers (e.g., neutrophils and IL-1β) was significantly higher in VAChT-KDHOM than wild-type mice. In contrast, LPS treatment resulted in a significant increase in lung’s α7 nicotinic receptor level in wild-type, but not in VAChT-KDHOM mice. However, treatment with PNU 282987, a selective α7 nicotinic receptor agonist, restored VAChT-KDHOM mice’s ability to increase α7 nicotinic receptor levels in response to LPS-induced acute lung injury and reduced lung inflammation. LPS also increased muscarinic receptors level in VAChT-KDHOM mice, and PNU 282987 treatment reduced this response. (4) Conclusions: Our data indicate that the anti-inflammatory effects of the lung cholinergic system involve an increase in the level of α7 nicotinic receptors. Pharmacological agents that increase the expression or the function of lung α7 nicotinic receptors have potential clinical uses for treating acute lung injury.
- ItemAcesso aberto (Open Access)Effects of VAChT reduction and α7nAChR stimulation by PNU-282987 in lung inflammation in a model of chronic allergic airway inflammation(Elsevier, 2020-09-05) Pinheiro, Nathalia Montouro; Miranda, Cláudia Jeane Claudino de Pontes; Santana, Fernanda Paula Roncon [UNIFESP]; Bittencourt-Mernak, Marcia Isabel [UNIFESP]; Arantes-Costas, Fernanda Magalhães; Olivo, Clarice Rosa; Perini, Adenir; Festa, Sergio [UNIFESP]; Caperuto, Luciana Chagas [UNIFESP]; Tiberio, Iolanda de Fátima Lopes Calvo; Prado, Marco Antônio Máximo; Martins, Milton de Arruda; Prado, Vânia Ferreira; Prado, Carla Máximo [UNIFESP]; http://lattes.cnpq.br/8960401765088965; Universidade Federal de São Paulo (UNIFESP)The cholinergic anti-inflammatory pathway has been shown to regulate lung inflammation and cytokine release in acute models of inflammation, mainly via α7 nicotinic receptor (α7nAChR). We aimed to evaluate the role of endogenous acetylcholine in chronic allergic airway inflammation in mice and the effects of therapeutic nAChR stimulation in this model. We first evaluated lung inflammation and remodeling on knock-down mice with 65% of vesicular acetylcholine transport (VAChT) gene reduction (KDVAChT) and wild-type(WT) controls that were subcutaneously sensitized and then inhaled with ovalbumin(OVA). We then evaluated the effects of PNU-282987(0.5-to-2mg/kg),(α7nAChR agonist) treatment in BALB/c male mice intraperitoneal sensitized and then inhaled with OVA. Another OVA-sensitized-group was treated with PNU-282987 plus Methyllycaconitine (MLA,1 mg/kg, α7nAChR antagonist) to confirm that the effects observed by PNU were due to α7nAChR. We showed that KDVAChT-OVA mice exhibit exacerbated airway inflammation when compared to WT-OVA mice. In BALB/c, PNU-282987 treatment reduced the number of eosinophils in the blood, BAL fluid, and around airways, and also decreased pulmonary levels of IL-4,IL-13,IL-17, and IgE in the serum of OVA-exposed mice. MLA pre-treatment abolished all the effects of PNU-282987. Additionally, we showed that PNU-282987 inhibited STAT3-phosphorylation and reduced SOCS3 expression in the lung. These data indicate that endogenous cholinergic tone is important to control allergic airway inflammation in a murine model. Moreover, α7nAChR is involved in the control of eosinophilic inflammation and airway remodeling, possibly via inhibition of STAT3/SOCS3 pathways. Together these data suggest that cholinergic anti-inflammatory system mainly α7nAChR should be further considered as a therapeutic target in asthma.