Resposta diferencial de células mononucleares do sangue periférico de indivíduos epiléticos e não epiléticos a estímulo inflamatório

• Ano de defesa: 2016
• Autor(a) principal: Flavia Mendes Amaral
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Epilepsia; Imunidade; Citocinas; Inflamação; Neurociências
• Link de acesso: http://hdl.handle.net/1843/BUOS-AJ4PHX

Introduction: Epilepsy is considered a strong predisposition for the generation of seizures, in the presence of cognitive and emotional dysfunction and is one of the most common neurological disorders worldwide, affecting 1% of the population. The role of inflammation in human epilepsy was first investigated by treatment with steroids and other anti-inflammatories, which showed an anticonvulsant effect in some epilepsy cases resistant to conventional treatment. Evidence suggests the activation of innate and adaptive immunity in human epilepsy, also suggesting that inflammation might contributeto the generation and recurrence of seizures and neuronal damage, on the other hand, recurring seizures may perpetuate the inflammatory response, leading to inflammation chronic. However, it is not clear whether the production of the inflammatory mediators by immune cells of patients with epilepsy and healthy subjects differ. Thus, in the presenthypothesis, we aimed to test the hypothesis that there is a deregulation in the intracelular signaling pathways in the peripheral mononuclear cells of patients with epilepsy, which contributes to a differential pattern in comparison with healthy subjects in response to an inflammatory stimulus. Methods: Experiments were performed in patients with Temporal Lobe Epilepsy and in healthy subjects, between 18 and 65 years old, who agreed to participate by signing the Informed Consent (IC). Individuals who agreed to participate in the study and who met the criteria established by the research underwent a brief medical history and collecting sociodemographic data, weight measurement and height, thequestionnaire application for assessment of anxiety and depressive symptoms (HAD) and collection the biological material (peripheral blood). Peripheral blood was collected in vacuum tubes containing sodium heparin and was performed Obtaining Mononuclear CellsPeripheral Blood protocol (PBMC). After counting cells, plating was performed and the cells were subjected to culture in the presence or absence of blocker of the enzymes PI3K, PI3Kg, mTOR and GSK3 for one hour in the CO2 incubator (37°C). After this brief incubation, PHA was added to stimulated wells or RPMI to wells without stimuli. The cellswere then incubated for 24 hours in the CO2 incubator. After incubation, the supernatant was separated from these cultures and were stored in a freezer at -20°C until the time of analysis. Dosages of cytokines and chemokines in the culture supernatant and plasma of patients with ELT and controls were performed by CBA method, according to manufacturer's instructions (BD Bioscience, San Diego, CA, USA). Kits were used for quantification of inflammatory proteins (Human Inflammation - IL-1, IL-6, IL-10, TNF and I L-12p70). Statistical analysis was performed using the statistical software Prism 5.0 (GraphPad, CA, USA) and data were presented as mean ± standard error of the mean(SEM). The level of significance was set at p <0.05. Results: Inhibition of all isoforms of PI3K or the selective inhibition PI3Kg, as mTOR inhibition increased levels of IL-6 and IL-8 only in unstimulated cell controls. Furthermore, the inhibitors do not significantly alter the levels of cells stimulated with PHA. Inhibition of all isoforms of PI3K or the selectiveinhibition PI3Kg, as well as inhibition of GSK3 and mTOR, resulted in a decrease in the levels of TNF and IL-10 in epileptic patients cells, stimulated with PHA, with the exception of inhibition for GSK3 in IL-10. PHA also demonstrated an increased IL-12p70 levels only in cells of epileptic patients and inhibition of PI3K and mTOR exacerbated the PHA effect only in the cells of patients. The selective inhibition of PI3K isoform did not affect the IL-12p70 levels. Conclusion: In this study was demonstrated that cytokine production by immune cells from patients with ELT is controlled differently in cells from healthy controls. This differential regulation may be dependent on the activity of different intracellular molecules such as PI3K, mTOR and GSK-3. This different response to inflammatory stimuli and the alteration in the intracellular signaling pathways could contribute to the physiopathogenesis of epilepsies.