Evaluation of cytokine production by J774.G8 macrophages after treatment with biotherapics


  • Camila Siqueira
  • Diogo Kuczera
  • Eneida Da Lozzo
  • Dorly Buchi
  • José Nelson Couceiro
  • Carla Holandino


cytokines, macrophages, biotherapics


Introduction: Strains of macrophages, such as murine J774.G8 macrophages, are susceptible to influenza A infection [1]. One of the responses to viral infection involves the production of various types of immunostimulatory cytokines by infected cells [2]. Methods: In the present study, the macrophage strain J774.G8, maintained in RPMI medium, was submitted to treatment with 10% V/V of two different biotherapics prepared from influenza H3N2, both at 30x. Additionally, two control groups were analyzed: macrophages stimulated with water 30x and macrophages without any treatment. Biotherapics were prepared from intact H3N2 influenza virus and H3N2 inactivated by alcohol 70%. The compounding of both biotherapics followed this procedure: one part of viral particles was diluted in 9 parts of sterile distilled water. The 1:10 sample was submitted to 100 mechanical succussions using Autic® Brazilian machine, originating the first dilution, named decimal (1x). 1 ml of this solution was diluted in 9 ml of solvent and was submitted to 100 succussions, generating biotherapic 2x. This procedure was successively repeated, according to Brazilian Homeopathic Pharmacopoeia, to obtain the biotherapic 30x. By the same technique, water vehicle was prepared in the potency of 30x to be used as control. All samples were prepared under sterile and aseptic conditions, using laminar flow cabinet, class II, and were stored in the refrigerator (8ºC), to avoid microbiological contamination. J774.G8 macrophages were stimulated for 2 days, in a total of six stimuli. Immediately before infection with 25 µl of H3N2 influenza virus, the supernatants were collected and frozen at -20 ºC for later analysis. Next, 24 hours after the virus infection, the supernatants were aliquoted and frozen under the same conditions. Three independent experiments were done in triplicate. Analysis of supernatants was performed by flow cytometry using the Mouse Inflammation Kit. The cytokines detected in this experiment were IL-10, IL 12, TNF-α and MCP1. Results: In all cases, there were no significant differences compared to control groups. However, the production of TNF-α detected in macrophages treated by intact and inactivated biotherapics presented a tendency to increase after infection. In fact, similar results were previously detected in other experiments conducted only with the intact biotherapic [3]. The release of the cytokine MCP1 in all experimental situations presented a tendency to decrease after the viral infection when compared to untreated macrophages. No statistically significant difference was detected in the production of IL 12 and IL 10. These experiments will be repeated to confirm the data obtained.




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