Isolamento, purificação e caracterização parcial do fator quimiotático de neutrófilos liberado pela Leishmania infantum em cultura

Abstract

The complex of diseases that comprehends Leishmaniasis is still a public health issue in many areas of the world with more than 1 million cases of all clinical forms reported yearly. There is a lot to be understood how the parasites interact with the mammalian cell host, including neutrophil and monocyte derived cells. Previous studies reported that neutrophils were the first cells to migrate towards the site of Leishmania infection. Indeed, neutrophils can actually help the progression of Leishmania infection, because they act as “shield cells” decreasing the capability of other defense cells to detect the parasites. The objectives of this study are to identify, purify and characterize molecules that are released by Leishmania promastigotes in culture which present potential chemotactic features for neutrophils. In order to characterize this chemotactic factor, L. infantum was cultured in HOMEM medium for 144 h and the supernatant was collected (MLP cond) and treated with different temperatures, trypsin, size exclusion. Molecules smaller than 10 kDa showed activity compared to the positive control (MLP cond), when the size fractionated samples were pulled back together, there was a higher activity. In addition, fractions treated at 100ºC for 10 min had a 2-fold increase in activity compared to no heat treatment. Samples were treated with trypsin and trypsin inhibitor still remained with the same chemotactic activity (P>0.05, in one way ANOVA statistical analysis). These results suggested that the chemotaxis can be due to more than one molecule of different sizes. FPLC was also used to purify the chemotactic factor. An anionic exchanger column (HiTrap Capto DEAE 1mL, General Electric) was used and the pH of operation was set at 8.0. The fractions were collected and tested for activity, but inconclusive results were obtained. As the research is still in progress, future steps were suggested to achieve the objectives.