Constitutive nitric oxide synthase-like enzyme in two species involved in cutaneous and mucocutaneous leishmaniasis.

Leishmania is an obligate intracellular parasite that primarily inhabits macrophages. The destruction of the parasite in the host cell is a fundamental mechanism for infection control. In addition, inhibition of the leishmanicidal activity of macrophages seems to be related to the ability of some species to inhibit the production of nitric oxide (NO) by depleting arginine. Some species of Leishmania have the ability to produce NO from a constitutive nitric oxide synthase-like enzyme (cNOS-like). However, the localization of cNOS-like in Leishmania has not been described before. As such, this study was designed to locate cNOS-like enzyme and NO production in promastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. NO production was initially quantified by flow cytometry, which indicated a significant difference in NO production between L. (L.) amazonensis (GMFC = 92.17 +/- 4.6) and L. (V.) braziliensis (GMFC = 18.89 +/- 2.29) (P < 0.05). Analysis of cNOS expression by immunoblotting showed more expression in L. (L.) amazonensis versus L. (V.) braziliensis. Subsequently, cNOS-like immunolabeling was observed in promastigotes in regions near vesicles, the flagellar pocket and mitochondria, and small clusters of particles appeared to be fusing with vesicles suggestive of glycosomes, peroxisome-like-organelles that compartmentalize the glycolytic pathway in trypanosomatid parasites. In addition, confocal microscopy analysis demonstrated colocalization of cNOS-like and GAPDH, a specific marker for glycosomes. Thus, L. (L.) amazonensis produces greater amounts of NO than L. (V.) braziliensis, and both species present the cNOS-like enzyme inside glycosomes.

Hierarchical Cluster Analysis of Three-Dimensional Reconstructions of Unbiased Sampled Microglia Shows not Continuous Morphological Changes from Stage 1 to 2 after Multiple Dengue Infections in Callithrix penicillata.

It is known that microglial morphology and function are related, but few studies have explored the subtleties of microglial morphological changes in response to specific pathogens. In the present report we quantitated microglia morphological changes in a monkey model of dengue disease with virus CNS invasion. To mimic multiple infections that usually occur in endemic areas, where higher dengue infection incidence and abundant mosquito vectors carrying different serotypes coexist, subjects received once a week subcutaneous injections of DENV3 (genotype III)-infected culture supernatant followed 24 h later by an injection of anti-DENV2 antibody. Control animals received either weekly anti-DENV2 antibodies, or no injections. Brain sections were immunolabeled for DENV3 antigens and IBA-1. Random and systematic microglial samples were taken from the polymorphic layer of dentate gyrus for 3-D reconstructions, where we found intense immunostaining for TNFα and DENV3 virus antigens. We submitted all bi- or multimodal morphological parameters of microglia to hierarchical cluster analysis and found two major morphological phenotypes designated types I and II. Compared to type I (stage 1), type II microglia were more complex; displaying higher number of nodes, processes and trees and larger surface area and volumes (stage 2). Type II microglia were found only in infected monkeys, whereas type I microglia was found in both control and infected subjects. Hierarchical cluster analysis of morphological parameters of 3-D reconstructions of random and systematic selected samples in control and ADE dengue infected monkeys suggests that microglia morphological changes from stage 1 to stage 2 may not be continuous.