Avaliação dos efeitos do TNF-alfa humano em Schistosoma mansoni
Data
2021
Autores
Lopes Junior, Ednilson Hilário [Unifesp]
Orientadores
Santos, Katia Cristina Pereira Oliveira [Unifesp]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Caracterizar os efeitos in vitro do tratamento com TNF-α humano na ovoposição, nos níveis de lactato, glicose e ATP e avaliar os níveis de expressão dos mRNAs do receptor de TNF (SmTNFR), lactato desidrogenase (LDH) e as quatro proteínas transportadoras de glicose (SGTPs) em S. mansoni. Métodos: Casais de vermes adultos de S. mansoni foram tratados in vitro com diferentes doses de hTNF-α (5, 20 e 40 ng/mL), em paralelo ao respectivo controle negativo por 5 dias. Ao longo do tratamento, os ovos foram contados diariamente, o meio de cultura e os casais de vermes foram coletados no 1º, 3º e 5º dia de tratamento e armazenados para análise posterior. As dosagens de lactato e glicose foram realizadas diretamente no meio de cultura (método colorimétrico), ATP foi dosado diretamente no lisado do parasita (detecção de quimioluminescência) e a expressão gênica foi quantificada por PCR em tempo real utilizando mRNA extraído dos vermes adultos tratados. Os esquistossômulos foram tratados e o ATP foi quantificado nas mesmas condições que os vermes adultos . Resultados: Notou-se um aumento significativo na contagem de ovos em 40ng/mL no 2º dia, uma diminuição em 20 e 40ng/mL no 3º dia e uma diminuição em 5 e 40ng/mL no 4º dia. Além disso, observou-se que o total de ovos não se altera ao longo dos 5 dias de tratamento com hTNF-α, porém a dinâmica da ovoposição está aumentada de maneira dose dependente, quanto maior a dose de tratamento mais rapidamente ocorre a ovoposição. Foi detectada uma diminuição na produção de lactato no tratamento com 5 e 20ng/mL no 3º e 5º dia, bem como uma diminuição na captação de glicose a 40ng/mL no 1º e 3º dia. Em relação ao ATP, verificou-se um aumento em sua produção a 40ng/mL no 5º dia para os casais de vermes adultos e nenhuma alteração em esquistossômulo. Não se observou nenhuma mudança de expressão nos níveis de mRNA dos genes testados. Conclusão: O hTNF-α não afeta a fecundidade do parasita, mas influencia a dinâmica da postura dos ovos; a produção de lactato, ATP e a captação de glicose são relevantes para a regulação desse evento e também sofrem alterações frente ao tratamento com a citocina humana. Neste trabalho, foi demonstrado que a interação molecular entre o hospedeiro e o parasita é relevante para a regulação fina do metabolismo e a fisiologia da ovoposição do S. mansoni e a sua importância para o sucesso da adaptação desta relação ecológica e para o parasitismo.
Objective: To characterize in vitro effects of treatment with human TNF-alpha on egg-laying, levels of lactate, glucose and ATP and to evaluate the mRNA's expression levels of TNF receptor (SmTNFR), lactate dehydrogenase (LDH) and four glucose transporter proteins (SGTPs) in S. mansoni. Methods: Couples of adult worms of S. mansoni were in vitro treated with different doses of hTNF-alpha (5, 20 and 40ng/mL), in parallel to the respective negative control for 5 days. Throughout the treatment, the eggs were counted daily, the culture medium and the worm pairs were collected on the 1st, 3rd, and 5th days of treatment and stored for later analysis. The lactate and glucose measurements were performed directly in the culture medium (colorimetric method), ATP was directly measured in the parasite lysate (detection of chemiluminescence) and the gene expression was quantified by real-time PCR using mRNA extracted from the treated adult worms. Schistosomula were treated and ATP was quantified in the same conditions of adult worms. Results: A significant increase in egg count was observed by 40ng/mL on the 2nd day, a decrease by 20 and 40ng/mL on the 3rd day, and a decrease by 5 and 40ng/mL on the 4th day. Furthermore, the total number of eggs does not change over the 5 days of hTNF-alpha treatment, however, the egg-laying dynamic is increased in a dose-dependent manner, the higher the treatment dose, the faster the egg-laying occurs. A decrease in the lactate production was detected by the treatment with 5 and 20ng/mL on the 3rd and 5th day. A decrease in glucose uptake was also detected by the treatment with 40ng/mL on the 1st and 3rd days. In relation to ATP, there was an increase in its production at 40ng/mL on the 5th day for couples of adult worms and no changes in schistosomula. No expression changes were detected in the mRNA levels of tested genes. Conclusion: hTNF-α does not affect the fertility of the parasite worm, but it does influence the dynamics of egg-laying; the production of lactate, ATP, and the uptake of glucose may be relevant for the regulation of this event and undergo changes in face of treatment with the human cytokine. In this work, it was demonstrated that the molecular interaction between the host and the parasite is relevant to the fine regulation of the metabolism and egg-laying physiology of S. mansoni and its importance for the successful adaptation of this ecological relationship and for parasitism.
Objective: To characterize in vitro effects of treatment with human TNF-alpha on egg-laying, levels of lactate, glucose and ATP and to evaluate the mRNA's expression levels of TNF receptor (SmTNFR), lactate dehydrogenase (LDH) and four glucose transporter proteins (SGTPs) in S. mansoni. Methods: Couples of adult worms of S. mansoni were in vitro treated with different doses of hTNF-alpha (5, 20 and 40ng/mL), in parallel to the respective negative control for 5 days. Throughout the treatment, the eggs were counted daily, the culture medium and the worm pairs were collected on the 1st, 3rd, and 5th days of treatment and stored for later analysis. The lactate and glucose measurements were performed directly in the culture medium (colorimetric method), ATP was directly measured in the parasite lysate (detection of chemiluminescence) and the gene expression was quantified by real-time PCR using mRNA extracted from the treated adult worms. Schistosomula were treated and ATP was quantified in the same conditions of adult worms. Results: A significant increase in egg count was observed by 40ng/mL on the 2nd day, a decrease by 20 and 40ng/mL on the 3rd day, and a decrease by 5 and 40ng/mL on the 4th day. Furthermore, the total number of eggs does not change over the 5 days of hTNF-alpha treatment, however, the egg-laying dynamic is increased in a dose-dependent manner, the higher the treatment dose, the faster the egg-laying occurs. A decrease in the lactate production was detected by the treatment with 5 and 20ng/mL on the 3rd and 5th day. A decrease in glucose uptake was also detected by the treatment with 40ng/mL on the 1st and 3rd days. In relation to ATP, there was an increase in its production at 40ng/mL on the 5th day for couples of adult worms and no changes in schistosomula. No expression changes were detected in the mRNA levels of tested genes. Conclusion: hTNF-α does not affect the fertility of the parasite worm, but it does influence the dynamics of egg-laying; the production of lactate, ATP, and the uptake of glucose may be relevant for the regulation of this event and undergo changes in face of treatment with the human cytokine. In this work, it was demonstrated that the molecular interaction between the host and the parasite is relevant to the fine regulation of the metabolism and egg-laying physiology of S. mansoni and its importance for the successful adaptation of this ecological relationship and for parasitism.
Descrição
Citação
Lopes Junior, Ednilson Hilário. Avaliação dos efeitos do TNF-alfa humano em Schistosoma mansoni. 2021. 92 f. Dissertação (Mestrado em Microbiologia e Imunologia) – Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (Unifesp), São Paulo, 2021.