Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response.

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106 cells per well on 24-well plates in order to obtain 2 x 105 macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Recent advances in the development and clinical application of miRNAs in infectious diseases.

In the search for new biomarkers and therapeutic targets for infectious diseases, several molecules have been investigated. Small RNAs, known as microRNAs (miRs), are important regulators of gene expression, and have emerged as promising candidates for these purposes. MiRs are a class of small, endogenous non-coding RNAs that play critical roles in several human diseases, including host-pathogen interaction mechanisms. Recently, miRs signatures have been reported in different infectious diseases, opening new perspectives for molecular diagnosis and therapy. MiR profiles can discriminate between healthy individuals and patients, as well as distinguish different disease stages. Furthermore, the possibility of assessing miRs in biological fluids, such as serum and whole blood, renders these molecules feasible for the development of new non-invasive diagnostic and prognostic tools. In this manuscript, we will comprehensively describe miRs as biomarkers and therapeutic targets in infectious diseases and explore how they can contribute to the advance of existing and new tools. Additionally, we will discuss different miR analysis platforms to understand the obstacles and advances of this molecular approach and propose their potential clinical applications and contributions to public health.