Diana Moreira

Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species.

Leishmania infantum modulates host macrophage mitochondrial metabolism by hijacking the SIRT1-AMPK axis

Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.

Leishmania-Infected MHC Class IIhigh Dendritic Cells Polarize CD4+ T Cells toward a Nonprotective T-bet+ IFN-γ+ IL-10+ Phenotype

A differential behavior among infected and bystander dendritic cells (DCs) has been explored in different infection models. We have analyzed both populations sorted on contact with visceral Leishmania infantum on a susceptible mice model evaluating the subsequent repercussions on adaptive immune response. Our results demonstrate a clear dichotomy between the immunomodulatory abilities of bystander and infected DCs. The bystander population presents increased levels of IL-12p40 and costimulatory molecules being capable to induce CD4+ T cell activation with immune protective capabilities. In contrast, infected DCs, which express lower costimulatory molecules and higher levels of IL-10, promote the development of Leishmania Ag-specific, nonprotective T-bet+IFN-γ+IL-10+ CD4+ T cells with an effector phenotype. This specific polarization was found to be dependent on IL-12p70. Splenic infected DCs recovered from chronic infected animals are similarly capable to polarize ex vivo syngeneic naive CD4+ T cells toward a T-bet+IFN-γ+IL-10+ phenotype. Further analysis revealed that only MHC class IIhigh–infected DCs were responsible for this polarization. The adoptive transfer of such polarized CD4+ T cells facilitates visceral leishmaniasis in BALB/c mice in a clear contrast with their counterpart generated with bystander DCs that significantly potentiate protection. Further, we demonstrated that CD4+ T cells primed by infected DCs in an IL-10 free system, thus deprived of T-bet+IFN-γ+IL-10+ population, restore the immune response and reduce parasite load, supporting a deleterious role of IFN-γ+IL-10+ T cells in the maintenance of infection. Overall, our results highlight novel subversion mechanisms by which nonprotective T-bet+IFN-γ+IL-10+ T cells are associated with chronicity and prolonged parasite persistence.

The Warburg effect in mycobacterial granulomas is dependent on the recruitment and activation of macrophages by interferon-γ

Granulomas are the hallmark of mycobacterial disease. Here, we demonstrate that both the cell recruitment and the increased glucose consumption in granulomatous infiltrates during Mycobacterium avium infection are highly dependent on interferon‐γ (IFN‐γ). Mycobacterium avium‐infected mice lacking IFN‐γ signalling failed to developed significant inflammatory infiltrations and lacked the characteristic uptake of the glucose analogue fluorine‐18‐fluorodeoxyglucose (FDG). To assess the role of macrophages in glucose uptake we infected mice with a selective impairment of IFN‐γ signalling in the macrophage lineage (MIIG mice). Although only a partial reduction of the granulomatous areas was observed in infected MIIG mice, the insensitivity of macrophages to IFN‐γ reduced the accumulation of FDG. In vivo, ex vivo and in vitro assays showed that macrophage activated by IFN‐γ displayed increased rates of glucose uptake and in vitro studies showed also that they had increased lactate production and increased expression of key glycolytic enzymes. Overall, our results show that the activation of macrophages by IFN‐γ is responsible for the Warburg effect observed in organs infected with M. avium.

The impact of distinct culture media in Leishmania infantum biology and infectivity

An ideal culture medium for Leishmania promastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinct in vitro and in vivo infectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for the in vitro maintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

Data concerning the psychometric properties of the Behavioral Inhibition/Behavioral Activation Scales for the Portuguese population.

The behavioral inhibition/behavioral activation (BIS/BAS) scales (Carver & White, 1994), which allow rating the Grays motivational systems, were translated and adapted into Portuguese. In this study, the authors present the procedure and the psychometric analyses of the Portuguese version of the scales, which included basic item and scales psychometric characteristics, as well as confirmatory and exploratory factor analyses. After the psychometric analyses provided evidence for the quality of the Portuguese version of the scales, the normative data was provided by age and school grade. The confirmatory factor analysis of the BIS/BAS scales that the authors performed did not demonstrate satisfactory fit for the 2- or 4-factor solution. The authors also tested the more recent 5-factor model, but the fit indices remained inadequate. As fit indices were not satisfactory they proceeded with an exploratory factor analysis to examine the structure of the Portuguese scales. These psychometric analyses provided evidence of a successful translation of the original scales. Therefore these scales can now be used in future research with Portuguese or Brazilian population.

ADHD, CD, and ODD: Systematic review of genetic and environmental risk factors

This review aims to analyze the relationships between Attention-Deficit Hyperactivity Disorder (ADHD), Oppositional Defiant Disorder (ODD), and Conduct Disorder (CD), particularly regarding the relative importance of genetic and environmental factors in the development of these disorders. Studies that examined at least two of these disorders were obtained from multiple databases, following the procedures of the Cochrane Collaboration initiative. Of the 279 documents obtained, nine were retained for in-depth analysis and were considered eligible for inclusion. In addition, eight studies from the manual search were included. The objectives, methodological aspects (sample and instruments), and the main conclusions were extracted from each study. Overall, the results suggest that (a) the causes for the onset and maintenance of these disorders are more associated with genetic factors than environmental factors, although the importance of the latter is recognized, and (b) children with ADHD have a predisposition to manifest behaviors that are common to ODD and CD, including the antisocial behavior that these children often display.

Psychiatric monitoring of not guilty by reason of insanity outpatients

Individuals deemed Not Guilty by Reason of Insanity (NGRI) by the courts, under Article 20 of the Portuguese Criminal Code, have often committed very serious crimes. It is unreasonable to consider that these patients were usually kept without adequate supervision after the security measure had been declared extinct. They often decompensated after leaving the institution where they complied with the security measure, and/or relapsed to alcohol and drug abuse. Very often, severe repeated crime erupted again. Considering this, there was an urgent need to keep a follow-up assessment of these patients in order to prevent them from relapsing in crime. This work presents the results of a psychiatric follow-up project with NGRI outpatients. The main goals of the project were: ensuring follow-up and appropriate therapeutic responses for these patients, maintaining all individuals in a care network, and preventing them from decompensating. The team consisted of a psychiatrist, a nurse, and a psychologist. Seventy-two patients were monitored during two years. Results demonstrated the unequivocal need to follow up decompensated patients after the court order is extinguished. Suggestions are presented for a better framing and psychiatric follow-up of these patients.

AMPK in Pathogens

During host-pathogen interactions, a complex web of events is crucial for the outcome of infection. Pathogen recognition triggers powerful cellular signaling events that is translated into the induction and maintenance of innate and adaptive host immunity against infection. In opposition, pathogens employ active mechanisms to manipulate host cell regulatory pathways toward their proliferation and survival. Among these, subversion of host cell energy metabolism by pathogens is currently recognized to play an important role in microbial growth and persistence. Extensive studies have documented the role of AMP-activated protein kinase (AMPK) signaling, a central cellular hub involved in the regulation of energy homeostasis, in host-pathogen interactions. Here, we highlight the most recent advances detailing how pathogens hijack cellular metabolism by suppressing or increasing the activity of the host energy sensor AMPK. We also address the role of lower eukaryote AMPK orthologues in the adaptive process to the host microenvironment and their contribution for pathogen survival, differentiation, and growth. Finally, we review the effects of pharmacological or genetic AMPK modulation on pathogen growth and persistence.

Metabolic Crosstalk Between Host and Parasitic Pathogens

A complex network that embraces parasite-host intrinsic factors and the microenvironment regulated the interaction between a parasite and its host. Nutritional pressures exerted by both elements of this duet thus dictate this host-parasite niche. To survive and proliferate inside a host and a harsh nutritional environment, the parasites modulate different nutrient sensing pathways to subvert host metabolic pathways. Such mechanism is able to change the flux of distinct nutrients/metabolites diverting them to be used by the parasites. Apart from this nutritional strategy, the scavenging of nutrients, particularly host fatty acids, constitutes a critical mechanism to fulfil parasite nutritional requirements, ultimately defining the host metabolic landscape. The host metabolic alterations that result from host-parasite metabolic coupling can certainly be considered important targets to improve diagnosis and also for the development of future therapies. Metabolism is in fact considered a key element within this complex interaction, its modulation being crucial to dictate the final infection outcome.