Immunology

Chronic Kidney Disease (CKD) patients present a micro inflammation state due to failure renal function. The calcitriol has been described as an anti-inflammatory factor that might modulates the inflammatory response in CKD patients. However, these patients have deficiency of Calcitriol due to failure renal function. But, synthesis of this vitamin has been reported in extra renal production, as in monocytes. In this context, it has been reported that the supplementation with 25 vitamin D (calcidiol or inactive form of vitamin D) induces monocytes to downregulate inflammation, due to the intracellular 1α-hidroxilase that converts calcidiol to calcitriol in these cells. Besides some reports used RT-qPCR, Western Blot or immunofluorescence techniques to investigate the expression of inflammatory and vitamin D machinery biomarkers in several disease, in the present study we used flow cytometry technique to evaluate the effect of 25 vitamin D on CD14, Toll-like receptor 4 (TLR4), vitamin D receptor (VDR), 1-α hydroxylase (CYP27), 24 hydroxylase (CYP24) in monocytes lineage (U937). The U937 culture was incubated with healthy or CKD serum and treatment with/without 25-vitamin D (50 ng/ml for 24 h) to evaluate CD14, TRL4, VDR, CYP27 and CYP24 expression. This protocol showed the advantage to investigate the effect of treatment with 25 vitamin D on the intracellular and cell membrane biomarkers expression quickly and simultaneously. In addition, this technique is not laborious, but easy to perform and to interpret compared to RT-qPCR, western blot or immunofluorescence.

In malaria, rosetting phenomenon is a condition where a Plasmodium-infected erythrocyte stably adheres to at least an uninfected erythrocyte. This phenomenon that occurs in all species of human malaria parasite is likely to be an immune escape mechanism for the parasite. However, it has been associated with malaria pathogenesis, possibly by facilitating microvasculature occlusion along with direct endothelial cytoadherence by the infected erythrocytes. There are different microscopy-based techniques to visualize rosettes but neither of these techniques has yet to qualify as the official “gold standard” method. We have found that these techniques can be used interchangeably, provided that the conditions of the experiments are properly controlled. Here, we presented three methods as options for rosetting assay, i.e., the unstained wet mount technique, acridine orange based-fluorescence microscopy technique and Giemsa stained wet mount method, with preparation steps that enable consistent performance in rosetting experiments.

Leishmaniasis is a parasitic disease caused by the obligatory intracellular protozoa Leishmania spp. Current therapeutic options are limited and thus, drug discovery against leishmaniasis is very important. Nevertheless, there is a great difficulty to develop therapeutic strategies against the disease because the parasite deploys various mechanisms to evade the immune system and multiply inside the host. Among the main factors of the immunity that are recruited to confront the Leishmania infection are the macrophages (MΦs) that produce effector molecules such as Nitric Oxide (NO) and Reactive Oxygen Species (ROS). Therefore, efficient drug agents should combine the antileishmanial effect of these gaseous transmitters along with the enhancement of the host’s adaptive immunity. In the quest of therapeutic alternatives, natural products have been extensively studied and are considered as candidate antileishmanial agents since they exhibit specific properties in modulating the host’s immune response towards an effective anti-leishmanial cell-mediated immunity capable to eliminate parasitic dissemination. In the current protocol, Leishmania-infected MΦs (J774A.1 cell line) that have been treated with various increasing concentrations of a natural compound, are tested for the production of the aforementioned molecules. In order to detect NO production, we employ the Griess colorimetric nitrite assay and quantification relies on the construction of an accurate standard curve using appropriate standards of known concentration. ROS detection and quantification is achieved by flow cytometry and relies on the use of carboxy-H₂DCFDA, an indicator that converts to a fluorescent form when interacts with ROS molecules.