Joana Cunha

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.

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.

Characterization of the biology and infectivity of Leishmania infantum viscerotropic and dermotropic strains isolated from HIV+ and HIV- patients in the murine model of visceral leishmaniasis

BackgroundLeishmaniasis is a group of diseases with a variety of clinical manifestations. The form of the disease is highly dependent on the infective Leishmania species and the immunological status of the host. The infectivity of the parasite strain also plays an important role in the progression of the infection. The aim of this work is to understand the influence of the natural infectivity of Leishmania strains in the outcome of visceral leishmaniasis.MethodsIn this study we have characterized four strains of L. infantum in terms of molecular typing, in vitro cultivation and differentiation. Two strains were isolated from HIV+ patients with visceral leishmaniasis (Bibiano and E390M), one strain was isolated from a cutaneous lesion in an immunocompetent patient (HL) and another internal reference strain causative of visceral leishmaniasis (ST) also from an immunocompetent patient was used for comparison. For this objective, we have compared their virulence by in vitro and in vivo infectivity in a murine model of visceral leishmaniasis.ResultsMolecular typing unraveled a new k26 sequence attributed to MON-284 zymodeme and allowed the generation of a molecular signature for the identification of each strain. In vitro cultivation enabled the production of promastigotes with comparable growth curves and metacyclogenesis development. The HL strain was the most infective, showing the highest parasite loads in vitro that were corroborated with the in vivo assays, 6 weeks post-infection in BALB/c mice. The two strains isolated from HIV+ patients, both belonging to two different zymodemes, revealed different kinetics of infection.ConclusionDifferences in in vitro and in vivo infectivity found in the murine model were then attributed to intrinsic characteristics of each strain. This work is supported by other studies that present the parasite’s inherent features as factors for the multiplicity of clinical manifestations and severity of leishmaniasis.

Testing thermal comfort of trekking boots: An objective and subjective evaluation

The study of the thermal comfort of the feet when using a specific type of shoe is of paramount importance, in particular if the main goal of the study is to attend to the needs of users. The main aim of this study was to propose a test battery for thermal comfort analysis and to apply it to the analysis of trekking boots. Methodologically, the project involves both objective and subjective evaluations. An objective evaluation of the thermal properties of the fabrics used in the boots was developed and applied. In addition, the thermal comfort provided when using the boots was also assessed both subjective and objectively. The evaluation of the thermal comfort during use, which was simulated in a laboratory environment, included the measurement of the temperature and moisture of the feet. The subjective assessment was performed using a questionnaire. From the results obtained, it was possible to define an optimal combination of fabrics to apply to trekking boots by considering the provided thermal insulation, air permeability and wicking. The results also revealed that the subjective perception of thermal comfort appears to be more related to the increase in temperature of the feet than to the moisture retention inside the boot. Although the evaluation of knits used in the boots indicated that a particular combination of fibres was optimal for use in the inner layer, the subjective and objective evaluation of thermal comfort revealed that the evaluation provided by users did not necessarily match the technical assessment data. No correlation was observed between the general comfort and specific thermal comfort assessments. Finally, the identification of thermal discomfort by specific foot areas would be useful in the process of designing and developing boots.

Protein Malnutrition Impairs the Immune Response and Influences the Severity of Infection in a Hamster Model of Chronic Visceral Leishmaniasis

Leishmaniasis remains one of the worlds most devastating neglected tropical diseases. It mainly affects developing countries, where it often co-exists with chronic malnutrition, one of the main risk factors for developing the disease. Few studies have been published, however, on the relationship between leishmaniasis progression and malnutrition. The present paper reports the influence of protein malnutrition on the immune response and visceral disease development in adult hamsters infected with Leishmania infantum fed either standard or low protein diets. The low protein diet induced severe malnutrition in these animals, and upon infection with L. infantum 33% had severe visceral leishmaniasis compared to only 8% of animals fed the standard diet. The infected, malnourished animals showed notable leukocyte depletion, mild specific antibody responses, impairment of lymphoproliferation, presence of parasites in blood (16.67% of the hamsters) and significant increase of the splenic parasite burden. Animals fed standard diet suffered agranulocytosis and monocytopenia, but showed stronger specific immune responses and had lower parasite loads than their malnourished counterparts. The present results show that protein malnutrition promotes visceral leishmaniasis and provide clues regarding the mechanisms underlying the impairment of the immune system.

Crucial CD8+ T-lymphocyte cytotoxic role in amphotericin B nanospheres efficacy against experimental visceral leishmaniasis

UNLABELLED This work aims to develop poly(d,l-lactide-co-glycolide) (PLGA)-nanospheres containing amphotericin B (AmB) with suitable physicochemical properties and anti-parasitic activity for visceral leishmaniasis (VL) therapy. When compared with unloaded-PLGA-nanospheres, the AmB-loaded PLGA-nanospheres displayed an increased particle size without affecting the polydispersity and its negative surface charge. AmB stability in the PLGA-nanospheres was >90% over 60-days at 30°C. The AmB-PLGA-nanospheres demonstrated significant in vitro and in vivo efficacy and preferential accumulation in the visceral organs. In addition, an immune-modulatory effect was observed in mice treated with AmB-PLGA-nanospheres, correlating with improved treatment efficacy. The in vitro cytotoxic response of the T-lymphocytes revealed that AmB-PLGA-nanospheres efficacy against VL infection was strictly due to the action of CD8(+)- but not CD4(+)-T lymphocytes. Overall, we demonstrate a crucial role for CD8(+) cytotoxic T lymphocytes in the efficacy of AmB-PLGA nanospheres, which could represent a potent and affordable alternative for VL therapy. FROM THE CLINICAL EDITOR This study demonstrates a crucial role for CD8+ T lymphocytes in eliminating visceral leishmaniasis in a murine model by enhancing the cytotoxic efficacy of CD8+ T-cells via amphotericin-B-PLGA nanospheres, paving a way to a unique, potentially more potent and cost-effective therapeutic strategy.

Serological evaluation of experimentally infected dogs by LicTXNPx-ELISA and amastigote-flow cytometry.

Canine visceral leishmaniasis (CVL) is characterized by a high incidence of asymptomatic infections. Because of the high prevalence of asymptomatic dogs in the endemic areas of visceral leishmaniasis (VL), a sensitive test is required for an accurate diagnosis. In this study, we evaluated the detection of symptomatic and asymptomatic Leishmania infantum infection in dogs using the secreted LicTXNPx antigen (Leishmania infantum cytosolic tryparedoxin peroxidase) in an ELISA format and compared it to soluble Leishmania antigens from promastigote or amastigote forms (SPLA and SALA) and two other unrelated secreted Leishmania proteins (LiTXN1 and TDR1). Moreover, we evaluated the diagnostic potential using the promastigote or amastigote-flow cytometric methodologies. The assays utilized sera collected from a cohort of L. infantum experimentally infected dogs, in which the intravenous or intradermal parasite injection mimics a symptomatic or asymptomatic pattern of infection, respectively. Our study indicated that anti-LicTXNPx antibodies were present in both symptomatic and asymptomatic experimental infections. Among the different Leishmania recombinant proteins tested, LicTXNPx showed a good predictive correlation with total soluble promastigote or amastigote Leishmania antigens, suggesting this antigen as a good candidate for a marker in either symptomatic or asymptomatic infection. The use of flow cytometry using both forms of live parasites was also tested with the same group of dogs. Amastigotes were shown to have more advantages than promastigotes for the serological diagnostic in both symptomatic and asymptomatic dogs, since higher continuous levels of anti-amastigote antibodies were detected during the course of experimental infection. Moreover, additional studies were done using sera from non-infected dogs and clinically asymptomatic and symptomatic dogs with confirmed naturally occurring L. infantum infections. The sensitivities of amastigote and promastigote flow cytometry were 96% vs. 89%, respectively, while the specificity for both was 93.2%. Therefore, our findings showed for the first time the potential of amastigote-flow cytometry regarding their applicability to detect both symptomatic and asymptomatic VL canine infections.

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.

Exploring geometric morphology in shape memory textiles: design of dynamic light filters

Thermo-responsive Shape Memory Alloys are able to adopt a temporary configuration and return to their programmed shape when heated to a determined activation temperature. The possibility to integrate them in textile substrates creates potential to develop smart textiles whose shape change explores functional and expressive purposes. The aim of this research is to develop shape memory woven textiles in which dynamic behavior achieves predefined geometric shapes. The requirement of geometric morphology was addressed through origami techniques. Combining foldability properties with shape change, it is possible to design textile structures with a variable number of layers. Difference in light transmittance is analyzed according to layer variation. Experiments conducted explore methodological processes aimed at future developments in dynamic light filters research. The results highlight a process to design textiles with predefined geometric morphologies that can be activated electrically, and delineate a further study in order to improve the shape memory textile behavior.

Detection of human neutrophil elastase (HNE) on wound dressings as marker of inflammation

Chronic wound fluids have elevated concentration of human neutrophil elastase (HNE) which can be used as inflammation/infection marker. Our goal is to develop functional materials for fast diagnosis of wound inflammation/infection by using HNE as a specific marker. For that, fluorogenic peptides with a HNE-specific cleavage sequence were incorporated into traditional textile dressings, to allow real-time detection of the wound status. Two different fluorogenic approaches were studied in terms of intensity of the signal generated upon HNE addition: a fluorophore 7-amino-4-trifluormethylcoumarin (AFC) conjugated to a HNE-specific peptide and two fluorophore/quencher pairs (FAM/Dabcyl and EDANS/Dabcyl) coupled to a similar peptide as a Förster resonance energy transfer (FRET) strategy. Also, two immobilization methods were tested: sonochemistry immobilization onto a cotton bandage and glutaraldehyde (GTA)-assisted chemical crosslinking onto a polyamide dressing. The immobilized fluorogenic AFC peptide showed an intense fluorescence emission in the presence of HNE. HNE also induced an enhanced fluorescent signal with the EDANS/Dabcyl FRET peptide which showed to be a more sensitive and effective strategy than the AFC peptide. However, its chemical immobilization onto the polyamide dressing greatly decreased its detection, mainly due to the more difficult access of the enzyme to the cleavage sequence of the immobilized peptide. After optimization of the in situ immobilization, it will be possible to use these fluorescence-functionalized dressings for an effective and specific monitoring of chronic wounds by simply using a portable ultraviolet (UV) light source. We envision that the development of this point-of-care medical device for wound control will have a great impact on patient’s life quality and reduction of costs on health care system.