Use of antimicrobial peptides and its nanogel formulations in the treatment of leishmaniasis

Parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease endemic in many developing countries as well as in the Mediterranean area. Current treatments are inefficient, associated with high toxicity, severe side effects and most importantly, the high costs associated to the treatment are far from suitable for developing countries. In this sense, there is an urgent need to find new drugs and a new drug delivery system to treat leishmaniasis. Several studies have shown that antimicrobial peptides (AMPs), components of our immune system, are able to help the organism resist to the invasion of some pathogens, including Leishmania, by having a direct antimicrobial function as well as a capacity for immunomodulation. Their specificity to microorganisms and the low probability to develop resistance, make them very good candidates for the development of a new drug formulation. Our proposal during this project is to increase the solubility and bioavailability of selected AMPs, described in the literature as having an active effect against Leishmania. We showed that hyaluronic acid (HA) derivative nanogel has an ability to entrap hydrophobic peptides. HA is naturally present in vertebrate organisms and its viscoelastic properties, biodegradability, biocompatibility and absence of immunogenicity, make it suitable for pharmaceutical and medical applications

Paracoccidioides brasiliensis: estudo do ciclo sexual

Dissertação de mestrado em Genética MolecularThe thermodimorphic fungal pathogen Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, one of the most prevalent systemic mycoses endemic in Latin America, occurring mainly in Brazil, Colombia and Venezuela. The morphological transformation of P. brasiliensis is characterized by the existence of two different morphological forms: a mycelium/conidial form that is present at environmental temperatures (below 25°C); and a multiple budding yeast form, present at temperatures of the mammalian host (37°C). The sexual cycle in P. brasiliensis has not been observed in nature or laboratory conditions. Nevertheless, in the present study, we detected low expression levels of mating-related genes, such as α-pheromone, α and a-pheromone receptors (PREB and PREA), and heterothallic mating loci (MAT1-1 and MAT1-2), in yeast and mycelial forms, and verified that heterothallic strains of opposite mating-types are able to express α- pheromone, and both pheromone receptors. In order to further evaluate the functional activity of mating-related genes, particularly α-pheromone and its cognate receptor (PreB), we took advantage of the heterologous expression of these P. brasiliensis genes in the corresponding Saccharomyces cerevisiae null mutants. Through several functional tests, including cell cycle arrest and shmoo formation, we showed that S. cerevisiae strains heterologously expressing PREB respond to synthetic α-pheromone of P. brasiliensis. In addition, mating ability of S. cerevisiae non-fertile strains was restored by the expression of PREB or α-pheromone in the corresponding null mutants. In general, this study demonstrates novel evidences for the existence of a functional mating signaling system in P. brasiliensis.O fungo patogénico termodimórfico Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose, uma das mais prevalentes micoses sistémicas, endémica da América Latina, ocorrendo principalmente no Brasil, Colômbia e Venezuela. A transformação morfológica de P. brasiliensis é caracterizada pela existência de duas formas distintas: a temperaturas ambientais (25°C) existe sob a forma de micélio/conídeo; e a temperaturas do hospedeiro (37°C) sob forma de levedura. Apesar de ainda não ter sido observado a existência de ciclo sexual em P. brasiliensis, no presente estudo detetámos níveis de expressão de genes relacionados com a reprodução sexuada em diversos fungo, tais como, feromona-α, recetor da feromona α e a (PREB e PREA) e ainda o MAT locus (MAT1-1 e MAT1-2). Verificámos que estirpes heterotálicas, de tipos de acasalamento opostos, têm a capacidade de expressar a feromona-α e ambos os recetores. De forma a avaliar a atividade funcional de genes relacionados com a reprodução sexuada, particularmente a feromona-α e respetivo recetor (PreB), procedemos à expressão destes genes de P. brasiliensis em estirpes mutantes de Saccharomyces cerevisiae. Através de vários testes funcionais, incluindo paragem de ciclo celular e formação de shmoos, mostrámos que a estirpe de S. cerevisiae que expressa PREB tem a capacidade de responder à feromona-α sintética de P. brasiliensis. Para além disso, a capacidade de acasalamento de estirpes de S. cerevisiae não férteis foi restabelecida pela expressão heteróloga da feromona-α e PREB. Este estudo demonstra novas evidências para a existência de um sistema de sinalização de acasalamento funcional em P. brasiliensis

Functionality of the paracoccidioides mating α-pheromone-receptor system

Recent evidence suggests that Paracoccidioides species have the potential to undergo sexual reproduction, although no sexual cycle has been identified either in nature or under laboratory conditions. In the present work we detected low expression levels of the heterothallic MAT loci genes MAT1-1 and MAT1-2, the a-pheromone (PBa) gene, and the a- and apheromone receptor (PREB and PREA) genes in yeast and mycelia forms of several Paracoccidioides isolates. None of the genes were expressed in a mating type dependent manner. Stimulation of P. brasiliensis MAT1-2 strains with the synthetic a pheromone peptide failed to elicit transcriptional activation of MAT1-2, PREB or STE12, suggesting that the strains tested are insensitive to a-pheromone. In order to further evaluate the biological functionality of the pair a-pheromone and its receptor, we took advantage of the heterologous expression of these Paracoccidioides genes in the corresponding S. cerevisiae null mutants. We show that S. cerevisiae strains heterologously expressing PREB respond to Pba pheromone either isolated from Paracoccidioides culture supernatants or in its synthetic form, both by shmoo formation and by growth and cell cycle arrests. This allowed us to conclude that Paracoccidioides species secrete an active a-pheromone into the culture medium that is able to activate its cognate receptor. Moreover, expression of PREB or PBa in the corresponding null mutants of S. cerevisiae restored mating in these non-fertile strains. Taken together, our data demonstrate pheromone signaling activation by the Paracoccidioides a-pheromone through its receptor in this yeast model, which provides novel evidence for the existence of a functional mating signaling system in Paracoccidioides.MHJS and JFM were supported by Fundacão para a Ciência e Tecnologia (FCT) grants. This work was supported by a grant from FCT (PTDC/BIA-MIC/ 108309/2008)

Breast cancer targeted photothermal therapy mediated by hyaluronic acid functionalized reduced graphene oxide

The use of graphene-based nanomaterials in cancer photothermal therapy (PTT) is an emerging alternative to the currently available cancer treatments. In this regard, reduced graphene oxide (rGO) has been widely explored for cancer PTT due to its excellent photothermal capacity. However, rGO has some limitations, such as low colloidal stability and water insolubility, as well as absence of targeting capacity towards cancer cells. Herein, rGO produced by an environmentally- friendly method was functionalized with an amphiphilic polymer based on hyaluronic acid (HA-rGO) through hydrophobic-hydrophobic interactions for application in targeted breast cancer PTT. The functionalization improved rGO colloidal stability and cytocompatibility towards normal and breast cancer cells, as well as conferred targeting capacity towards CD44 overexpressing breast cancer cells. In addition, the photothermal effect mediated by HA-rGO upon laser irradiation reduced breast cancer cells’ viability. Overall, HA-rGO demonstrated a great potential for being used on-demand and selective treatment of breast cancer cells.Rita Lima-Sousa and Cátia G. Alves acknowledge funding from the grant UBI Santander/Totta.info:eu-repo/semantics/publishedVersio

Writing 3D In vitro models of human tendon within a biomimetic fibrillar support platform

Tendinopathies are poorly understood diseases for which treatment remains challenging. Relevant in vitro models to study human tendon physiology and pathophysiology are therefore highly needed. Here we propose the automated 3D writing of tendon microphysiological systems (MPSs) embedded in a biomimetic fibrillar support platform based on cellulose nanocrystals (CNCs) self-assembly. Tendon decellularized extracellular matrix (dECM) was used to formulate bioinks that closely recapitulate the biochemical signature of tendon niche. A monoculture system recreating the cellular patterns and phenotype of the tendon core was first developed and characterized. This system was then incorporated with a vascular compartment to study the crosstalk between the two cell populations. The combined biophysical and biochemical cues of the printed pattern and dECM hydrogel were revealed to be effective in inducing human-adipose-derived stem cells (hASCs) differentiation toward the tenogenic lineage. In the multicellular system, chemotactic effects promoted endothelial cells migration toward the direction of the tendon core compartment, while the established cellular crosstalk boosted hASCs tenogenesis, emulating the tendon development stages. Overall, the proposed concept is a promising strategy for the automated fabrication of humanized organotypic tendon-on-chip models that will be a valuable new tool for the study of tendon physiology and pathogenesis mechanisms and for testing new tendinopathy treatments.The authors thank Hospital da Prelada (Porto, Portugal) for providing adipose tissue samples. The authors acknowledge the financial support from Project NORTE-01-0145-FEDER 000021 supported by Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), the European Union Framework Program for Research and Innovation HORIZON 2020, under the Twinning Grant Agreement 810850-Achilles, and European Research Council Grant Agreement 772817 and 101069302, Fundação para a Ciência e a Tecnologia for the Ph.D. grant PD/BD/129403/2017 (to S.M.B.) financed through the doctoral program in Tissue Engineering, Regenerative Medicine and Stem Cells (TERM&SC), for Contract 2020.03410.CEECIND and 2022.05526.PTDC (to R.M.A.D.). The authors acknowledge Doctor Alberto Pardo for performing the rheology measurements of the PL bioink. The schematics in Figures 1, 2A, 4A, and 6A and Table of Contents graphic were created with BioRender.com

Immunomodulation From Moderate Exercise Promotes Control of Experimental Cutaneous Leishmaniasis

Physical exercise has been described as an important tool in the prevention and treatment of numerous diseases as it promotes a range of responses and adaptations in several biological systems, including the immune system. Studies on the effect of exercise on the immune system could play a critical role in improving public health. Current literature suggests that moderate intensity exercise can modulate the Th1/Th2 dichotomy directing the immune system to a Th1 cellular immune response, which favors the resolution of infections caused by intracellular microorganisms. Leishmaniasis is a group of diseases presenting a wide spectrum of clinical manifestations that range from self-limiting lesions to visceral injuries whose severity can lead to death. The etiological agents responsible for this group of diseases are protozoa of the genus Leishmania. Infections by the parasite Leishmania major in mice (Balb/c) provide a prototype model for the polarization of CD4+ T cell responses of both Th1 (resistance) or Th2 (susceptibility), which determines the progression of infections. The aim of this study was to evaluate the effect of exercise on the development of L. major experimental infections by scanning the pattern of immune response caused by exercise. Groups of Balb/c mice infected with L. major were divided into groups that preformed a physical exercise of swimming three times a week or were sedentary along with treatment or not with the reference drug, meglumine antimoniate. Animals in groups submitted to physical exercise did not appear to develop lesions and presented a significantly lower parasite load independent of drug treatment. They also showed a positive delayed hypersensitivity response to a specific Leishmania antigen compared to control animals. The IFN-γ/IL-4 and IFN-γ/IL10 ratios in trained animals were clearly tilted to a Th1 response in lymph node cells. These data suggest that moderate intensity exercise is able to modulate the Th1 response that provides a protective effect against the development of leishmanial lesions

Emissions of putative isoprene oxidation products from mango branches under abiotic stress

Although several per cent of net carbon assimilation can be re-released as isoprene emissions to the atmosphere by many tropical plants, much uncertainty remains regarding its biological significance. In a previous study, we detected emissions of isoprene and its oxidation products methyl vinyl ketone (MVK) and methacrolein (MACR) from tropical plants under high temperature/light stress, suggesting that isoprene is oxidized not only in the atmosphere but also within plants. However, a comprehensive analysis of the suite of isoprene oxidation products in plants has not been performed and production relationships with environmental stress have not been described. In this study, putative isoprene oxidation products from mango (Mangifera indica) branches under abiotic stress were first identified. High temperature/light and freeze-thaw treatments verified direct emissions of the isoprene oxidation products MVK and MACR together with the first observations of 3-methyl furan (3-MF) and 2-methyl-3-buten-2-ol (MBO) as putative novel isoprene oxidation products. Mechanical wounding also stimulated emissions of MVK and MACR. Photosynthesis under 13CO2 resulted in rapid (<30min) labelling of up to five carbon atoms of isoprene, with a similar labelling pattern observed in the putative oxidation products. These observations highlight the need to investigate further the mechanisms of isoprene oxidation within plants under stress and its biological and atmospheric significance. © 2013 The Author

Ag85-focused T-cell immune response controls Mycobacterium avium chronic infection

CD4+ T cells are essential players for the control of mycobacterial infections. Several mycobacterial antigens have been identified for eliciting a relevant CD4+ T cell mediated-immune response, and numerous studies explored this issue in the context of Mycobacterium tuberculosis infection. Antigen 85 (Ag85), a highly conserved protein across Mycobacterium species, is secreted at the early phase of M. tuberculosis infection leading to the proliferation of Ag85-specific CD4+ T cells. However, in the context of Mycobacterium avium infection, little is known about the expression of this antigen and the elicited immune response. In the current work, we investigated if a T cell receptor (TCR) repertoire mostly, but not exclusively, directed at Ag85 is sufficient to mount a protective immune response against M. avium. We show that P25 mice, whose majority of T cells express a transgenic TCR specific for Ag85, control M. avium infection at the same level as wild type (WT) mice up to 20 weeks post-infection (wpi). During M. avium infection, Ag85 antigen is easily detected in the liver of 20 wpi mice by immunohistochemistry. In spite of the propensity of P25 CD4+ T cells to produce higher amounts of interferon-gamma (IFNγ) upon ex vivo stimulation, no differences in serum IFNγ levels are detected in P25 compared to WT mice, nor enhanced immunopathology is detected in P25 mice. These results indicate that a T cell response dominated by Ag85-specific T cells is appropriate to control M. avium infection with no signs of immunopathology.This work was developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Fellowships from the Portuguese Foundation for Science and Technoloy (FCT) were attributed to BCR (SFRH/BD/80352/2011; QREN-POPH through the Fundo Social Europeu (FSE) and national funds from MEC] and to CN (SFRH/BPD/112001/2015; POPH through FSE and national funds from MCTES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

TLR9 activation dampens the early inflammatory response to paracoccidioides brasiliensis, Impacting host survival

Background: Paracoccidioides brasiliensis causes paracoccidioidomycosis, one of the most prevalent systemic mycosis in Latin America. Thus, understanding the characteristics of the protective immune response to P. brasiliensis is of interest, as it may reveal targets for disease control. The initiation of the immune response relies on the activation of pattern recognition receptors, among which are TLRs. Both TLR2 and TLR4 have been implicated in the recognition of P. brasiliensis and regulation of the immune response. However, the role of TLR9 during the infection by this fungus remains unclear.J.F. Menino was supported by a grant from Fundacao para a Ciencia e Tecnologia (FCT), Portugal (SFRH/BD/33446/2008). This work was supported by a grant from FCT (PTDC/BIA-MIC/108309/2008). M. Saraiva is a Ciencia 2007 fellow and M. Sturme is a Ciencia 2008 fellow. We would also like to thank FAPESP (Fundacao para Amparo a Pesquisa do Estado de Sao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript