Replication of Rocio virus in primary cultures of mouse neural cells

Instituto Evandro Chagas/ Ministry of Health and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal do Pará. Belém, PA, Brazil.Universidade Federal do Pará. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Ciência, Tecnologia, Inovação e Insumos Estratégicos. Instituto Evandro Chagas. Ananindeua, PA, Brasil.This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. Methods: Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. Results: Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. Conclusion: The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system

Emergence of new immunopathogenic factors in human yellow fever: polarization of the M1/M2 macrophage response in the renal parenchyma

This research was funded by grant number 457664/2013-4 and 303999/2016-0.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade do Estado do Pará. Departamento de Patologia. Belém, PA, Brazil / Universidade de São Paulo. Faculdade de Medicina. São Paulo, SP, Brazil.Universidade do Estado do Pará. Departamento de Patologia. Belém, PA, Brazil.Universidade do Estado do Pará. Departamento de Patologia. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade de São Paulo. Faculdade de Medicina. São Paulo, SP, Brazil.Universidade do Estado do Pará. Departamento de Patologia. Belém, PA, Brazil.Universidade do Estado do Pará. Departamento de Patologia. Belém, PA, Brazil.Universidade do Estado do Pará. Departamento de Patologia. Belém, PA, Brazil / Universidade de São Paulo. Faculdade de Medicina. São Paulo, SP, Brazil / Universidade Federal do Pará. Núcleo de Medicina Tropical. Belém, PA, Brazil.Macrophages in the kidney play a pathogenic role in inflammation and fibrosis. Our study aimed to understand the polarisation of the M1 and M2 phenotypic profiles of macrophages in injured kidney tissue retrieved from fatal cases of yellow fever virus (YFV). A total of 11 renal tissue biopsies obtained from patients who died of yellow fever (YF) were analysed. To detect antibodies that promote the classical and alternative pathways of macrophage activation, immunohistochemical analysis was performed to detect CD163, CD68, inducible nitric oxide synthase (iNOS), arginase 1, interleukin (IL)-4, IL-10, interferon (IFN)-γ, IFN-β, tumour necrosis factor (TNF)-α, IL-13, and transforming growth factor (TGF)-β. There was a difference in the marker expression between fatal cases of YFV and control samples, with increased expression in the cortical region of the renal parenchyma. The immunoexpression of CD68 and CD163 receptors suggests the presence of activated macrophages migrating to infectious foci. The rise in IL-10, IL-4, and IL-13 indicated their potential role in the inactivation of the inflammatory macrophage response and phenotypic modulation of M2 macrophages. The altered expression of IFN-γ and IFN-β demonstrates the importance of the innate immune response in combating microorganisms. Our findings indicate that the polarisation of M1 and M2 macrophages plays a vital role in the renal immune response to YFV

Oral treatment with the extract of Euterpe oleracea Mart. improves motor dysfunction and reduces brain injury in rats subjected to ischemic stroke

PROPESP-UFPA.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. Centre for the Valorization of Amazonian Bioactive Compounds. Belém, PA, Brazil.Federal University of Pará. Centre for the Valorization of Amazonian Bioactive Compounds. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde e Ambiente. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Pará. Institute Biological Science. Laboratory of Pharmacology and Toxicology of Natural Products. Belém, PA, Brazil.Federal University of Pará. Institute Biological Science. Laboratory of Pharmacology and Toxicology of Natural Products. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Ischemic stroke is one of the principal causes of morbidity and mortality around the world. The pathophysiological mechanisms that lead to the formation of the stroke lesions range from the bioenergetic failure of the cells and the intense production of reactive oxygen species to neuroinflammation. The fruit of the açaí palm, Euterpe oleracea Mart. (EO), is consumed by traditional populations in the Brazilian Amazon region, and it is known to have antioxidant and anti-inflammatory properties. We evaluated whether the clarified extract of EO was capable of reducing the area of lesion and promoting neuronal survival following ischemic stroke in rats. Animals submitted to ischemic stroke and treated with EO extract presented a significant improvement in their neurological deficit from the ninth day onward. We also observed a reduction in the extent of the cerebral injury and the preservation of the neurons of the cortical layers. Taken together, our findings indicate that treatment with EO extract in the acute phase following a stroke can trigger signaling pathways that culminate in neuronal survival and promote the partial recovery of neurological scores. However, further detailed studies of the intracellular signaling pathways are needed to better understand the mechanisms involved

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications

Modelo in vitro de parkinsonismo experimental induzido por rotenona: investigação de mecanismos de ação, neuroproteção e morte celular

Increasing evidence has suggested a role for environmental factors, such as exposure to pesticides, in the pathogenesis of Parkinson’s disease. In experimental animals the exposure to rotenone, a common herbicide and piscicide, induces features of parkinsonism by inhibiting the activity of mitochondrial complex I. Here we propose to investigate rotenone-induced death of neurons by using primary neuron-enriched and neuron-glia cultures from the rat hippocampus and ventral mesencephalon. The neuronal loss was evaluated with the colorimetric MTT assay. Our results showed significant reduction in the cell viability after exposure to rotenone in a dose- but not in a timedependent manner. We also discovered a remarkable feature of rotenone-induced degeneration of cultured neurons. The higher susceptibility was observed in neuron-glia cultures from the ventral mesencephalon, suggesting that the presence of glia, especially microglia, is an important factor contributing to neurodegeneration. Also, as showed by immunohistochemistry, this type of culture presented the higher density of tirosinahidroxilase (TH)-positive neurons. Mechanistically, our results with calcium blockers showed a minimal role played by external calcium, and an important synergistic influence of the ions from the internal stores in the rotenone-induced neurodegeneration. Indeed, in this study, we report that aqueous extract of mahogany leaves didn’t protect against the rotenone-induced toxicity, in the used concentration; and promoted a synergistic effect when associated with rotenona. Finally, the mahogany leaves extract induced celular death both necrosis and apoptosis. The results of this study should advance our understanding of the mechanism of action for environmental factors in the pathogenesis of Parkinson’s disease.FAPESPA - Fundação Amazônia de Amparo a Estudos e PesquisasEvidências crescentes na literatura têm sugerido papel importante para os fatores ambientais, como a exposição a pesticidas, na patogênese da doença de Parkinson. Em animais experimentais, a exposição à rotenona, um pesticida e piscicida de uso comum, induz características de parkinsonismo através da inibição do complexo I mitocondrial. O objetivo deste estudo foi investigar a morte de neurônios induzida por rotenona utilizando culturas primárias mistas neurônio/glia derivadas de hipocampo e de mesencéfalo ventral de ratos, bem como o papel do Ca2+ na neste modelo experimental e a utilização de extrato aquoso de folhas de mogno com substâncias com alto poder antioxidante. A perda neuronal foi analisada com ensaios colorimétricos (MTT e LDH). Nossos resultados mostraram significativa redução na viabilidade celular após exposição à rotenona de maneira dependente de concentração, mas não dependente de tempo. Foi observada igual e elevada suscetibilidade em culturas mistas neurônio/glia derivadas de hipocampo e de mesencéfalo ventral ao agente neurotóxico. Em termos mecanicísticos, nossos resultados mostraram um papel discreto desempenhado pelo Ca2+ mitocondrial na neurodegeneração induzida por rotenona. Além disso, neste paradigma utilizado, verificamos que o extrato aquoso de folhas de mogno não promoveu proteção contra a toxicidade da rotenona, na concentração testada; ainda, promoveu efeito sinérgico em associação com rotenona. Verificou-se ainda que a rotenona, bem como o extrato de mogno promoveu indução de morte celular tanto por necrose quanto por apoptose, nas concentrações utilizadas. Os resultados deste estudo devem avançar nosso conhecimento sobre o mecanismo de ação de fatores ambientais na patogênese da doença de Parkinson

Acute and delayed biochemical, hematological, and neuromuscular responses to the Rest-pause resistance training method

Universidade Federal do Pará - Campus Universitário de Castanhal. Faculdade de Educação Física. Castanhal, PA, Brazil / Universidade Federal do Pará. Programa de Pós-Graduação em Ciências do Movimento Humano. Belém, PA, Brazil.Universidade Federal do Pará - Campus Universitário de Castanhal. Faculdade de Educação Física. Castanhal, PA, Brazil / Universidade Federal do Pará. Programa de Pós-Graduação em Ciências do Movimento Humano. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Universidade Federal do Pará. Núcleo de Estudos e Pesquisa em Lutas e Esportes de Combate. Belém, PA, Brazil.Universidade Federal do Pará - Campus Universitário de Castanhal. Faculdade de Educação Física. Castanhal, PA, Brazil / Universidade Federal do Pará. Programa de Pós-Graduação em Ciências do Movimento Humano. Belém, PA, Brazil.Objective: This investigation aimed to compare acute and delayed biochemical, hematological, and neuromuscular responses to the Rest-pause (RP) method and traditional resistance training (TRT) through 48 h. Methods: This crossover design study, in which the training sessions were separated by a 7-days washout interval. Twelve recreationally trained males (age: 22.4 ± 1.8 years) realized the RP protocol, consisting of two sets of 6-3-3 repetitions at 90% of 6-repetition maximum (6RM) and 20 s of rest between repetitions and 2:30 min between sets and TRT protocol, consistent of 3 sets of 8 repetitions at 85% of 6RM with 2 min between sets. Blood analyses and neuromuscular performance were measured before (PRE), immediately after (POST) and 24 (24H) and, 48 h (48H) after protocols. Results: Regarding between-moments comparisons, biochemical and hematological responses increased after protocols (p 0.05). Only creatine kinase, aspartate aminotransferase, neutrophiles, and white blood cells increased after protocols (p 0.05). No significant difference between moments was found for neuromuscular performance (p > 0.05). Regarding difference between groups, red blood cells and hemoglobin showed lower values for the RP in comparison to the TRT after 24H (p = 0.03). Conclusion: The sets configurations seem to be insufficient to induce an additional effect in muscle damage markers, immune responses, and neuromuscular performance. Finally, it seems that advanced techniques with lower volume don’t affect the immune system for long time periods

The GPER1 agonist G1 reduces brain injury and improves the qEEG and behavioral outcome of experimental ischemic stroke

PROPESP-UFPAFederal University of Pará. Joao de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. Joao de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. Joao de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. Joao de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. Joao de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde e Ambiente. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Pará. Joao de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. Biological Sciences Institute. Laboratory of Pharmacology and Toxicology of Natural Products. Belém, PA, Brazil.Federal University of Pará. Joao de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Stroke is one of the principal cerebrovascular diseases in human populations and contributes to a majority of the functional impairments in the elderly. Recent discoveries have led to the inclusion of electroencephalography (EEG) in the complementary prognostic evaluation of patients. The present study describes the EEG, behavioral, and histological changes that occur following cerebral ischemia associated with treatment by G1, a potent and selective G protein-coupled estrogen receptor 1 (GPER1) agonist in a rat model. Treatment with G1 attenuated the neurological deficits induced by ischemic stroke from the second day onward, and reduced areas of infarction. Treatment with G1 also improved the total brainwave power, as well as the theta and alpha wave activity, specifically, and restored the delta band power to levels similar to those observed in the controls. Treatment with G1 also attenuated the peaks of harmful activity observed in the EEG indices. These improvements in brainwave activity indicate that GPER1 plays a fundamental role in the mediation of cerebral injury and in the behavioral outcome of ischemic brain injuries, which points to treatment with G1 as a potential pharmacological strategy for the therapy of stroke

Increased relative delta bandpower and delta indices revealed by continuous qEEG monitoring in a rat model of ischemia-reperfusion

This work was funded in part by PROPESP-UFPA.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. Institute Biological Science. Laboratory of Pharmacology and Toxicology of Natural Products. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.Federal University of Pará. Institute Biological Science. Laboratory of Pharmacology and Toxicology of Natural Products. Belém, PA, Brazil.Federal University of Pará. João de Barros Barreto University Hospital. Laboratory of Experimental Neuropathology. Belém, PA, Brazil.The present study describes the electroencephalographic changes that occur during cerebral ischemia and reperfusion in animals submitted to transient focal cerebral ischemia by middle cerebral artery occlusion (MCAO) for 30 min. For this, male Wistar rats were divided into two groups (n = 6 animals/group): (1) sham (control) group, and (2) ischemic/reperfusion group. The quantitative electroencephalography (qEEG) was recorded during the ischemic and immediate reperfusion (acute) phases, and then once a day for 7 days after the MCAO (subacute phase). The acute phase was characterized by a marked increase in the relative delta wave band power (p < 0.001), with a smaller, but significant increase in the relative alpha wave bandpower in the ischemic stroke phase, in comparison with the control group (p = 0.0054). In the immediate reperfusion phase, however, there was an increase in the theta, alpha, and beta waves bandpower (p < 0.001), but no alteration in the delta waves (p = 0.9984), in comparison with the control group. We also observed high values in the delta/theta ratio (DTR), the delta/alpha ratio (DAR), and the (delta+theta)/(alpha+beta) ratio (DTABR) indices during the ischemia (p < 0.05), with a major reduction in the reperfusion phase. In the subacute phase, the activity of all the waves was lower than that of the control group (p < 0.05), although the DTR, DAR, and DTABR indices remained relatively high. In conclusion, early and accurate identification of decreased delta wave bandpower, DTR, DAR, and DTABR indices, and an increase in the activity of other waves in the immediate reperfusion phase may represent an important advance for the recognition of the effectiveness of reperfusion therapy

Emergence of New Immunopathogenic Factors in Human Yellow Fever: Polarisation of the M1/M2 Macrophage Response in the Renal Parenchyma

Macrophages in the kidney play a pathogenic role in inflammation and fibrosis. Our study aimed to understand the polarisation of the M1 and M2 phenotypic profiles of macrophages in injured kidney tissue retrieved from fatal cases of yellow fever virus (YFV). A total of 11 renal tissue biopsies obtained from patients who died of yellow fever (YF) were analysed. To detect antibodies that promote the classical and alternative pathways of macrophage activation, immunohistochemical analysis was performed to detect CD163, CD68, inducible nitric oxide synthase (iNOS), arginase 1, interleukin (IL)-4, IL-10, interferon (IFN)-γ, IFN-β, tumour necrosis factor (TNF)-α, IL-13, and transforming growth factor (TGF)-β. There was a difference in the marker expression between fatal cases of YFV and control samples, with increased expression in the cortical region of the renal parenchyma. The immunoexpression of CD68 and CD163 receptors suggests the presence of activated macrophages migrating to infectious foci. The rise in IL-10, IL-4, and IL-13 indicated their potential role in the inactivation of the inflammatory macrophage response and phenotypic modulation of M2 macrophages. The altered expression of IFN-γ and IFN-β demonstrates the importance of the innate immune response in combating microorganisms. Our findings indicate that the polarisation of M1 and M2 macrophages plays a vital role in the renal immune response to YFV

New Insights into the Mechanism of Immune-Mediated Tissue Injury in Yellow Fever: The Role of Immunopathological and Endothelial Alterations in the Human Lung Parenchyma

Yellow fever (YF) may cause lesions in different organs. There are no studies regarding the in situ immune response in the human lung and investigating immunopathological aspects in fatal cases can help to better understand the evolution of the infection. Lung tissue samples were collected from 10 fatal cases of human yellow fever and three flavivirus-negative controls who died of other causes and whose lung parenchymal architecture was preserved. In YFV-positive fatal cases, the main histopathological changes included the massive presence of diffuse alveolar inflammatory infiltrate, in addition to congestion and severe hemorrhage. The immunohistochemical analysis of tissues in the lung parenchyma showed significantly higher expression of E-selectin, P-selectin, ICAM-1, VCAM-1 in addition to cytokines such as IL-4, IL-10, IL-13, TNF- &alpha;, IFN-&gamma; and TGF-&beta; compared to the negative control. The increase in immunoglobulins ICAM-1 and VCAM-1 results in strengthening of tissue transmigration signaling. E-selectin and P-selectin actively participate in this process of cell migration and formation of the inflammatory infiltrate. IFN-&gamma; and TNF-&alpha; participate in the process of cell injury and viral clearance. The cytokines IL-4 and TGF-&beta;, acting in synergism, participate in the process of tissue regeneration and breakdown. The anti-inflammatory cytokines IL-4, IL-10 and IL-13 also act in the reduction of inflammation and tissue repair. Our study indicates that the activation of the endothelium aggravates the inflammatory response by inducing the expression of adhesion molecules and cytokines that contribute to the rolling, recruitment, migration and eliciting of the inflammatory process in the lung parenchyma, contributing to the fatal outcome of the disease