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Chronic neuroinflammation is characterized by increased blood-brain barrier (BBB) permeability, leading to molecular changes in the central nervous system that can be explored with biomarkers of active neuroinflammatory processes. Magnetic resonance imaging (MRI) has contributed to detecting lesions and permeability of the BBB. Ultra-small superparamagnetic particles of iron oxide (USPIO) are used as contrast agents to improve MRI observations. Therefore, we validate the interaction of peptide-88 with laminin, vectorized on USPIO, to explore BBB molecular alterations occurring during neuroinflammation as a potential tool for use in MRI. The specific labeling of NPS-P88 was verified in endothelial cells (hCMEC/D3) and astrocytes (T98G) under inflammation induced by interleukin 1ß (IL-1ß) for 3 and 24 hours. IL-1ß for 3 hours in hCMEC/D3 cells increased their co-localization with NPS-P88, compared with controls. At 24 hours, no significant differences were observed between groups. In T98G cells, NPS-P88 showed similar nonspecific labeling among treatments. These results indicate that NPS-P88 has a higher affinity towards brain endothelial cells than astrocytes under inflammation. This affinity decreases over time with reduced laminin expression. In vivo results suggest that following a 30-minute post-injection, there is an increased presence of NPS-P88 in the blood and brain, diminishing over time. Lastly, EAE animals displayed a significant accumulation of NPS-P88 in MRI, primarily in the cortex, attributed to inflammation and disruption of the BBB. Altogether, these results revealed NPS-P88 as a biomarker to evaluate changes in the BBB due to neuroinflammation by MRI in biological models targeting laminin.
Assuntos
Barreira Hematoencefálica , Laminina , Animais , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/metabolismo , Laminina/metabolismo , Doenças Neuroinflamatórias , Células Endoteliais/metabolismo , Inflamação/diagnóstico por imagem , Inflamação/metabolismo , Imageamento por Ressonância Magnética/métodosRESUMO
Chikungunya (CHIKV), Zika (ZIKV), and dengue viruses (DENV) are vector-borne pathogens that cause emerging and re-emerging epidemics throughout tropical and subtropical countries. The symptomatology is similar among these viruses and frequently co-circulates in the same areas, making the diagnosis arduous. Although there are different methods for detecting and quantifying pathogens, real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for detecting viruses. However, the currently developed assays frequently involve probes and high-cost reagents, limiting access in low-income countries. Therefore, this study aims to design and evaluate a quantitative one-step RT-qPCR assay to detect CHIKV, ZIKV, and DENV with high specificity, reproducibility, and low cost in multiple cell substrates. We established a DNA intercalating green dye-based RT-qPCR test that targets nsP1 of CHIKV, and NS5 gene of ZIKV, and DENV for the amplification reaction. The assay exhibited a high specificity confirmed by the melting curve analysis. No cross-reactivity was observed between the three viruses or unspecific amplification of host RNA. The sensitivity of the reaction was evaluated for each virus assay, getting a limit of detection of one RNA copy per virus. Standard curves were constructed, obtaining a reaction efficiency of ~ 100%, a correlation coefficient (R2) of ~ 0.97, and a slope of -3.3. The coefficient of variation (CV) ranged from 0.02 to 1.43. In addition, the method was optimized for viral quantification and tested in Vero, BHK-21, C6/36, LULO, and the Aedes cell lines. Thus, the DNA intercalating green dye-based RT-qPCR assay was a highly specific, sensitive, reproducible, and effective method for detecting and quantifying CHIKV, ZIKV, and DENV in different cell substrates that could also be applied in clinical samples.
Assuntos
Febre de Chikungunya , Vírus Chikungunya , Vírus da Dengue , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Infecção por Zika virus , Zika virus , Zika virus/genética , Zika virus/isolamento & purificação , Vírus Chikungunya/genética , Vírus Chikungunya/isolamento & purificação , Vírus da Dengue/genética , Vírus da Dengue/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Humanos , Febre de Chikungunya/diagnóstico , Febre de Chikungunya/virologia , Infecção por Zika virus/virologia , Infecção por Zika virus/diagnóstico , Dengue/virologia , Dengue/diagnóstico , Chlorocebus aethiops , Reprodutibilidade dos Testes , Células Vero , RNA Viral/genética , Linhagem CelularRESUMO
The chikungunya virus (CHIKV) has produced epidemic outbreaks of significant public health impact. The clinical symptoms of this disease are fever, polyarthralgia, and skin rash, generally self-limiting, although patients may develop a chronic disabling condition or suffer lethal complications. Unfortunately, there is no specific treatment or vaccine available. Thus, the search for effective therapies to control CHIKV infection is an urgent need. This study evaluated the antiviral activity of flavonoids isolated from Marcetia taxifolia by in vitro and in silico analysis. Cytotoxicity of compounds was determined by MTT assay and viral load was assessed in cell substrates supernatants by plaque-forming and RT-qPCR assays. Selected molecules were analyzed by molecular docking assays. Myricetin 3-rhamnoside (MR) and myricetin 3-(6-rhamnosylgalactoside) (MRG) were tested for antiviral assays and analyzed by the TCID50 method and RT-qPCR. MR exhibited dose-dependent antiviral activity, reducing viral titer at concentrations of 150-18.8 µg/mL by at least 1-log. Similarly, MRG showed a significant decrease in viral titer at concentrations of 37.5, 9.4, and 2.3 µg/mL. RT-qPCR analysis also displayed a substantial reduction of CHIKV RNA for both flavonoids. Furthermore, molecular docking of the selected flavonoids proposed the nsP3 macrodomain as a possible target of action. Our study reveals that MR and MRG could be considered promising anti-CHIKV therapeutic agents. Molecular modeling studies showed MR and MRG ligands with a high affinity for the N-terminal region of the nsP3 macrodomain, postulating them as a potential target of action for the CHIKV control.
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There is great interest on medium chain fatty acids (MCFA) for cardiovascular health. We explored the effects of MCFA on the expression of lipid metabolism and inflammatory genes in macrophages, and the extent to which they were mediated by the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPAR ß/δ). J774A.1 murine macrophages were exposed to octanoate or decanoate as MCFA, a long-chain fatty acid control (palmitate), or the PPAR ß/δ agonist GW501516, with or without lipopolysaccharide (LPS) stimulation, and with or without an siRNA-induced knockdown of PPAR ß/δ. MCFA increased the expression of Plin2, encoding a lipid-droplet associated protein with anti-inflammatory effects in macrophages, in a partially PPAR ß/δ-dependent manner. Both MCFA stimulated expression of the cholesterol efflux pump ABCA1, more pronouncedly under LPS stimulation and in the absence of PPAR ß/δ. Octanoate stimulated the expression of Pltp, encoding a phospholipid transfer protein that aids ABCA1 in cellular lipid efflux. Only palmitate increased expression of the proinflammatory genes Il6, Tnf, Nos2 and Mmp9. Non-stimulated macrophages exposed to MCFA showed less internalization of fluorescently labeled lipoproteins. MCFA influenced the transcriptional responses of macrophages favoring cholesterol efflux and a less inflammatory response compared to palmitate. These effects were partially mediated by PPAR ß/δ.
Assuntos
PPAR delta , PPAR beta , Camundongos , Animais , PPAR delta/metabolismo , PPAR beta/genética , PPAR beta/metabolismo , Caprilatos/farmacologia , Linhagem Celular , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Ácidos Graxos/farmacologia , Colesterol/metabolismo , Palmitatos/farmacologiaRESUMO
Laminin, a non-collagenous glycoprotein present in the brain extracellular matrix, helps to maintain blood-brain barrier (BBB) integrity and regulation. Neuroinflammation can compromise laminin structure and function, increasing BBB permeability. The aim of this paper is to determine if neuroinflammation-induced laminin functional changes may serve as a potential biomarker of alterations in the BBB. The 38 publications included evaluated neuroinflammation, BBB disruption, and laminin, and were assessed for quality and risk of bias (protocol registered in PROSPERO; CRD42020212547). We found that laminin may be a good indicator of BBB overall structural integrity, although changes in expression are dependent on the pathologic or experimental model used. In ischemic stroke, permanent vascular damage correlates with increased laminin expression (ß and γ subunits), while transient damage correlates with reduced laminin expression (α subunits). Laminin was reduced in traumatic brain injury and cerebral hemorrhage studies but increased in multiple sclerosis and status epilepticus studies. Despite these observations, there is limited knowledge about the role played by different subunits or isoforms (such as 411 or 511) of laminin in maintaining structural architecture of the BBB under neuroinflammation. Further studies may clarify this aspect and the possibility of using laminin as a biomarker in different pathologies, which have alterations in BBB function in common.
Assuntos
Barreira Hematoencefálica , Laminina , Biomarcadores/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Humanos , Laminina/metabolismo , Doenças NeuroinflamatóriasRESUMO
Mosquitoes are the most crucial insects in public health due to their vector capacity and competence to transmit pathogens, including arboviruses, bacterias and parasites. Re-emerging and emerging arboviral diseases, such as yellow fever virus (YFV), dengue virus (DENV), zika virus (ZIKV), and chikungunya virus (CHIKV), constitute one of the most critical health public concerns in Latin America. These diseases present a significant incidence within the human settlements increasing morbidity and mortality events. Likewise, among the different genus of mosquito vectors of arboviruses, those of the most significant medical importance corresponds to Aedes and Culex. In Latin America, the mosquito vector species of YFV, DENV, ZIKV, and CHIKV are mainly Aedes aegypti and Ae. Albopictus. Ae. aegypti is recognized as the primary vector in urban environments, whereas Ae. albopictus, recently introduced in the Americas, is more prone to rural settings. This minireview focuses on what is known about the epidemiological impact of mosquito-borne diseases in Latin American countries, with particular emphasis on YFV, DENV, ZIKV and CHIKV, vector mosquitoes, geographic distribution, and vector-arbovirus interactions. Besides, it was analyzed how climate change and social factors have influenced the spread of arboviruses and the control strategies developed against mosquitoes in this continent.
Assuntos
Infecções por Arbovirus/epidemiologia , Infecções por Arbovirus/prevenção & controle , Infecções por Arbovirus/virologia , Arbovírus , Doenças Transmitidas por Vetores/epidemiologia , Doenças Transmitidas por Vetores/prevenção & controle , Doenças Transmitidas por Vetores/virologia , Animais , Vírus Chikungunya , Clima , Culicidae/virologia , Vírus da Dengue , Interações entre Hospedeiro e Microrganismos , Humanos , América Latina/epidemiologia , Mosquitos Vetores , Saúde Pública , Fatores Sociais , Vírus da Febre Amarela , Zika virusRESUMO
Malnutrition is a risk factor for developing visceral leishmaniasis and its severe forms. Our group demonstrated that malnourished animals infected with Leishmania infantum had severe atrophies in lymphoid organs and T cell subpopulations as well as altered levels of thymic and splenic chemotactic factors, all of which resulted in dysfunctional lymphoid microenvironments that promoted parasite proliferation. Here, we hypothesize that malnutrition preceding parasite infection leads to structural and immunological changes in the gut mucosae, resulting in a failure in the immune response sensed in the intestine. To evaluate this, we analyzed the immunopathological events resulting from protein malnutrition in the guts of BALB/c mice infected with L. infantum. We observed lymphocytic/lymphoplasmacytic inflammatory infiltrates and lymphoid hyperplasia in the duodenum of well-nourished-infected mice; such alterations were worsened when malnutrition preceded infection. Parasite infection induced a significant increase of duodenal immunoglobulin A (IgA) of well-nourished animals, but those levels were significantly decreased in malnourished-infected mice. In addition, increased levels of Th17-related cytokines in duodenums of malnourished animals supported local inflammation. Together, our results suggest that the gut plays a potential role in responses to L. infantum infection-and that such responses are impaired in malnourished individuals.
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BACKGROUND AND AIM: Osteosarcoma (OSA) is the most common bone tumor in canines and humans. This study aimed to assess the cytotoxic and apoptotic effects of Colombian propolis samples on a canine OSA cell line (OSCA-8) by evaluating the expression of BCL-2, BAX, CASPASE 9, CASPASE 8, and TNFR1 genes involved in the apoptosis pathway. MATERIALS AND METHODS: After treating the cells with five Colombian propolis samples (Usm, Met, Fus, Sil, and Caj), we evaluated cell viability and lactate dehydrogenase (LDH) release. Early and late apoptosis was determined by flow cytometry using annexin V/propidium iodide. Furthermore, the effects of three selected samples on gene expression were analyzed by real-time polymerase chain reaction. RESULTS: The Colombian propolis samples reduced OSCA-8 cell viability and increased LDH release. All samples induced apoptosis significantly and upregulated BCL-2 and CASPASE 8 expression. Usm and Sil increased BAX expression, Met and Sil induced CASPASE 9 expression, and Usm increased TNFR1. CONCLUSION: Colombian propolis samples exhibited cytotoxic and apoptotic effects on canine OSA cells, and CASPASE 8 upregulation indicated apoptosis induction by the extrinsic pathway.
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Neurodegenerative diseases are characterized by increased permeability of the blood-brain barrier (BBB) due to alterations in cellular and structural components of the neurovascular unit, particularly in association with neuroinflammation. A previous screening study of peptide ligands to identify molecular alterations of the BBB in neuroinflammation by phage-display, revealed that phage clone 88 presented specific binding affinity to endothelial cells under inflammatory conditions in vivo and in vitro. Here, we aimed to identify the possible target receptor of the peptide ligand 88 expressed under inflammatory conditions. A cross-link test between phage-peptide-88 with IL-1ß-stimulated human hCMEC cells, followed by mass spectrometry analysis, was used to identify the target of peptide-88. We modeled the epitope-receptor molecular interaction between peptide-88 and its target by using docking simulations. Three proteins were selected as potential target candidates and tested in enzyme-linked immunosorbent assays with peptide-88: fibronectin, laminin subunit α5 and laminin subunit ß-1. Among them, only laminin subunit ß-1 presented measurable interaction with peptide-88. Peptide-88 showed specific interaction with laminin subunit ß-1, highlighting its importance as a potential biomarker of the laminin changes that may occur at the BBB endothelial cells under pathological inflammation conditions.
Assuntos
Barreira Hematoencefálica , Células Endoteliais/metabolismo , Inflamação/metabolismo , Laminina/metabolismo , Animais , Bacteriófago M13 , Biomarcadores , Células Cultivadas , Reagentes de Ligações Cruzadas , Fibronectinas/metabolismo , Ontologia Genética , Humanos , Interleucina-1beta/farmacologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , RatosRESUMO
Obesity and type 2 diabetes (T2D) are growing health problems associated with a loss of insulin sensitivity. Both conditions arise from a long-term energy imbalance, and frequently, lifestyle measures can be useful in its prevention, including physical activity and a healthy diet. Pancreatic ß-cells are determinant nutrient sensors that participate in energetic homeostasis needs. However, when pancreatic ß-cells are incapable of secreting enough insulin to counteract the reduced sensitivity, the pathology evolves to an insulin resistance condition. The primary nutrient that stimulates insulin secretion is glucose, but also, there are multiple dietary and hormonal factors influencing that response. Many studies of the physiology of ß-cells have highlighted the importance of glucose, fructose, amino acids, and free fatty acids on insulin secretion. The present review summarizes recent research on how ß-cells respond to the most abundant nutrients that influence insulin secretion. Taken together, understand the subjacent mechanisms of each nutrient on ß-cells can help to unravel the effects of mixed variables and complexity in the context of ß-cell pathology.