Cytochemical techniques and energy-filtering transmission electron microscopy applied to the study of parasitic protozoa

The study of parasitic protozoa plays a major role in cell biology, biochemistry and molecular biology. Numerous cytochemical techniques have been developed in order to unequivocally identify the nature of subcellular compartments. Enzyme and immuno-cytochemistry allow the detection of, respectively, enzymatic activity products and antigens in particular sites within the cell. Energy-filtering transmission electron microscopy permits the detection of specific elements within such compartments. These approaches are particularly useful for studies employing antimicrobial agents where cellular compartments may be destroyed or remarkably altered and thus hardly identified by standard methods of observation. In this regard cytochemical and spectroscopic techniques provide valuable data allowing the determination of the mechanisms of action of such compounds

DETC Induces Leishmania Parasite Killing in Human In Vitro and Murine In Vivo Models: A Promising Therapeutic Alternative in Leishmaniasis

Background: Chemotherapy remains the primary tool for treatment and control of human leishmaniasis. However, currently available drugs present serious problems regarding side-effects, variable efficacy, and cost. Affordable and less toxic drugs are urgently needed for leishmaniasis. Methodology/Principal Findings: We demonstrate, by microscopy and viability assays, that superoxide dismutase inhibitor diethyldithiocarbamate (DETC) dose-dependently induces parasite killing (p,0.001) and is able to ??????sterilize?????? Leishmania amazonensis infection at 2 mM in human macrophages in vitro. We also show that DETC-induced superoxide production (p,0.001) and parasite destruction (p,0.05) were reverted by the addition of the antioxidant N-acetylcysteine, indicating that DETC-induced killing occurs through oxidative damage. Furthermore, ultrastructural analysis by electron microscopy demonstrates a rapid and highly selective destruction of amastigotes in the phagosome upon DETC treatment, without any apparent damage to the host cell, including its mitochondria. In addition, DETC significantly induced parasite killing in Leishmania promastigotes in axenic culture. In murine macrophages infected with Leishmania braziliensis, DETC significantly induced in vitro superoxide production (p = 0.0049) and parasite killing (p = 0.0043). In vivo treatment with DETC in BALB/C mice infected with Leishmania braziliensis caused a significant decrease in lesion size (p,0.0001), paralleled by a 100-fold decrease (p = 0.0087) in parasite burden. Conclusions/Significance: Due to its strong leishmanicidal effect in human macrophages in vitro, its in vivo effectiveness in a murine model, and its previously demonstrated in vivo safety profile in HIV treatment, DETC treatment might be considered as a valuable therapeutic option in human leishmaniasis, including HIV/Leishmania co-infection

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field