Ana M. G. Silva

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.

Activation of Phosphatidylinositol 3-Kinase/Akt and Impairment of Nuclear Factor-κB: Molecular Mechanisms Behind the Arrested Maturation/Activation State of Leishmania infantum-Infected Dendritic Cells

Understanding the complex interactions between Leishmania and dendritic cells (DCs) is central to the modulation of the outcome of this infection, given that an effective immune response against Leishmania is dependent on the successful activation and maturation of DCs. We report here that Leishmania infantum promastigotes successfully infect mouse bone marrow-derived DCs without triggering maturation, as shown by a failure in the up-regulation of CD40 and CD86 expression, and that parasites strongly counteract the lipopolysaccharide-triggered maturation of DCs. A small increase in interleukin (IL)-12 and IL-10 transcription and secretion and a decrease in IL-6 were observed in infected cells. This arrested DC maturation state is actively promoted by parasites because heat-killed or fixed parasites increased cytokine and costimulatory molecule expression. At a molecular level, L. infantum rapidly induced activation of phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2, whereas no effect was observed in the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase proinflammatory pathways. Moreover, parasites actively promoted cleavage of the nuclear factor-κB p65(RelA) subunit, causing its impairment. The blockade of phosphatidylinositol 3-kinase/Akt by either treatment of bone marrow-derived DCs with wortmannin or transfection with an Akt dominant-negative mutant resulted in a strong decrease in infection rates, revealing for the first time a crucial role of this pathway on Leishmania engulfment by DCs. Overall, our data indicate that activation of Akt and impairment of nuclear factor-κB are responsible for immunogenicity subversion of L. infantum-infected DCs.

Quality of life of patients with congenital heart diseases

OBJECTIVES To assess the perception of the quality of life of adolescents and young adults with congenital heart disease and to examine the variables that have a negative impact on it and that add a resilience effect. METHODS A total of 22 male and 18 female patients, aged 12-26 years, of whom 27 were admitted to surgery and 13 were not, participated in this study. All patients had complete medical records and were interviewed once; demographic and clinical data were collected, and patients filled a questionnaire on quality of life, the WHOQOL-BREF, and underwent an interview on social support, educational style, self-image, functional limitations, and emotional adjustment. RESULTS/CONCLUSIONS Our patients showed a better perception of quality of life than did the general population, on the basis of psychological, social relationship and environment scales. Older patients hold a better perception of quality of life on the psychological scale. Cyanosis did not show any significant impact over perception of quality of life decay; however, the number of surgical procedures and the persistence of moderate-to-severe residual injuries had considerable detrimental effect. Social support had an impact on increasing resilience, promoting adjustment to illness. Several factors may play a role in adjustment to congenital heart disease, either improving the perception of quality of life or worsening it. We may conclude that some buffer variables on congenital heart disease may play roles in increasing the perception of quality of life of patients during their lifetime, social support probably explaining why the perception of quality of life is better than in the normal population. The number of surgeries and the moderate-to-severe residual injuries, however, reverted that effect.

Leishmania TDR1 structure, a unique trimeric glutathione transferase capable of deglutathionylation and antimonial prodrug activation

Thiol-dependent reductase I (TDR1), an enzyme found in parasitic Leishmania species and Trypanosoma cruzi, is implicated in deglutathionylation and activation of antimonial prodrugs used to treat leishmaniasis. The 2.3 Å resolution structure of TDR1 reveals a unique trimer of subunits each containing two glutathione-S-transferase (GST) domains. The similarities of individual domains and comparisons with GST classes suggest that TDR1 evolved by gene duplication, diversification, and gene fusion; a combination of events previously unknown in the GST protein superfamily and potentially explaining the distinctive enzyme properties of TDR1. The deglutathionylation activity of TDR1 implies that glutathione itself has regulatory intracellular roles in addition to being a precursor for trypanothione, the major low mass thiol present in trypanosomatids. We propose that activation of antiparasite Sb(V)-drugs is a legacy of the deglutathionylation activity of TDR1 and involves processing glutathione adducts with concomitant reduction of the metalloid to active Sb(III) species.

Metabolic variation during development in culture of Leishmania donovani promastigotes

The genome sequencing of several Leishmania species has provided immense amounts of data and allowed the prediction of the metabolic pathways potentially operating. Subsequent genetic and proteomic studies have identified stage-specific proteins and putative virulence factors but many aspects of the metabolic adaptations of Leishmania remain to be elucidated. In this study, we have used an untargeted metabolomics approach to analyze changes in the metabolite profile as promastigotes of L. donovani develop during in vitro cultures from logarithmic to stationary phase. The results show that the metabolomes of promastigotes on days 3–6 of culture differ significantly from each other, consistent with there being distinct developmental changes. Most notable were the structural changes in glycerophospholipids and increase in the abundance of sphingolipids and glycerolipids as cells progress from logarithmic to stationary phase.

Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism.

Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.

meso -Tetraarylporphyrins as dipolarophiles in 1,3-dipolar cycloaddition reactions

meso-Tetraarylporphyrins participate, as dipolarophiles, in 1,3-dipolar cycloaddition reactions with azomethine ylides to yield novel chlorins and isobacteriochlorins.

The contribution of Toll-like receptor 2 to the innate recognition of a Leishmania infantum silent information regulator 2 protein.

We have characterized a Leishmania protein belonging to the silent information regulator 2 (SIR2) family [SIR2 related protein 1 (SIR2RP1)] that might play an immunoregulatory role during infection through its capacity to trigger B‐cell effector functions. We report here that SIR2RP1 leads to the proliferation of activated B cells, causing increased expression of major histocompatibility complex (MHC) II and the costimulatory molecules CD40 and CD86, which are critical ligands for T‐cell cross‐talk during the development of adaptive immune responses. In contrast, B cells isolated from Toll‐like receptor 2 (TLR2) knockout mice were unable to respond to the SIR2RP1 stimulus. Similarly, SIR2RP1 induced the maturation of dendritic cells (DCs) in a TLR2‐dependent manner with the secretion of pro‐inflammatory cytokines [interleukin (IL)‐12 and tumour necrosis factor (TNF)‐α] and enhanced the costimulatory properties of DCs. Nevertheless, immunization assays demonstrated that TLR2‐deficient mice were able to mount a specific humoral response to SIR2RP1. Interestingly, further investigations showed that macrophages were activated by SIR2RP1 even in the absence of TLR2. Therefore, a different type of interplay between SIR2RP1 and the major antigen‐presenting cells in vivo could explain the immune response observed in TLR2‐deficient mice. Together, these results demonstrate that TLR2 signalling contributes to SIR2RP1 recognition by innate immune host cells.

Ohmic heating as a new efficient process for organic synthesis in water

A new and efficient process for organic synthesis in aqueous media based on a direct ohmic heating reactor is described. Four representative organic transformations, a Diels–Alder cycloaddition, a nucleophilic substitution, an N-alkylation and a Suzuki cross-coupling reaction, were performed using this process. The results, when compared with those obtained under conventional external heating (oil bath) and microwave heating, showed that ohmic reactor allows faster and more uniform heating and an induced increase of dynamics/mobility of charged species leading in several cases to higher reaction yields and shorter reaction times.

Characterization of Leishmania infantum thiol-dependent reductase 1 and evaluation of its potential to induce immune protection

The need to develop an effective vaccine against leishmaniasis to prevent the 2 million new cases each year led to the search for antigens able to elicit protection against infection with Leishmania. In this study, we have characterized a parasite‐specific protein of Leishmania infantum named thiol‐dependent reductase 1 (TDR1). The protein is present in both life cycle stages of L. infantum with a notable higher expression in the amastigote forms, suggesting a role in the interaction between the parasite and the mammalian host. Thiol‐dependent reductase 1 is localized in the cytosol, although we were able to detect the protein in the culture medium of both promastigotes and axenic amastigotes, and consequently, TDR1 is considered an excreted/secreted molecule of the parasite. Therefore, we have evaluated the potential of TDR1 recombinant protein to protect against experimental challenge with L. infantum parasites using a murine model. Despite a reduction in spleen parasite load in the chronic phase of disease, TDR1 administration was not effective in the protection of Balb/c mice against visceral leishmaniasis and thus TDR1 do not have a crucial role in the modulation of mammalian host immune response, as observed with its protein counterpart Tc52 of Trypanosoma cruzi.