Prevention of lipid droplet accumulation by DGAT1 inhibition ameliorates sepsis-induced liver injury and inflammation.

Background & Aims: Lipid droplet (LD) accumulation in cells and tissues is understood to be an evolutionarily conserved tissue tolerance mechanism to prevent lipotoxicity caused by excess lipids; however, the presence of excess LDs has been associated with numerous diseases. Sepsis triggers the reprogramming of lipid metabolism and LD accumulation in cells and tissues, including the liver. The functions and consequences of sepsis-triggered liver LD accumulation are not well known. Methods: Experimental sepsis was induced by CLP (caecal ligation and puncture) in mice. Markers of hepatic steatosis, liver injury, hepatic oxidative stress, and inflammation were analysed using a combination of functional, imaging, lipidomic, protein expression and immune-enzymatic assays. To prevent LD formation, mice were treated orally with A922500, a pharmacological inhibitor of DGAT1. Results: We identified that liver LD overload correlates with liver injury and sepsis severity. Moreover, the progression of steatosis from 24 h to 48 h post-CLP occurs in parallel with increased cytokine expression, inflammatory cell recruitment and oxidative stress. Lipidomic analysis of purified LDs demonstrated that sepsis leads LDs to harbour increased amounts of unsaturated fatty acids, mostly 18:1 and 18:2. An increased content of lipoperoxides within LDs was also observed. Conversely, the impairment of LD formation by inhibition of the DGAT1 enzyme reduces levels of hepatic inflammation and lipid peroxidation markers and ameliorates sepsis-induced liver injury. Conclusions: Our results indicate that sepsis triggers lipid metabolism alterations that culminate in increased liver LD accumulation. Increased LDs are associated with disease severity and liver injury. Moreover, inhibition of LD accumulation decreased the production of inflammatory mediators and lipid peroxidation while improving tissue function, suggesting that LDs contribute to the pathogenesis of liver injury triggered by sepsis. Impact and Implications: Sepsis is a complex life-threatening syndrome caused by dysregulated inflammatory and metabolic host responses to infection. The observation that lipid droplets may contribute to sepsis-associated organ injury by amplifying lipid peroxidation and inflammation provides a rationale for therapeutically targeting lipid droplets and lipid metabolism in sepsis.

Human neutrophil extracellular traps do not impair in vitro Toxoplasma gondii infection.

Introduction: Toxoplasma gondii, responsible for causing toxoplasmosis, is a prevalent food and waterborne pathogen worldwide. It commonly infects warm-blooded animals and affects more than a third of the global human population. Once ingested, the parasite enters the host's small intestine and rapidly disseminates throughout the body via the bloodstream, infiltrating various tissues. Leukocyte-driven responses are vital against T. gondii, with neutrophils playing a dual role: swiftly recruited to infection sites, releasing inflammatory mediators, and serving as a replication hub and Trojan horses, aiding parasite spread. Neutrophils from various hosts release extracellular traps (NETs) against the protozoan. However, gaps persist regarding the mechanisms of NETs production to parasite and their significance in infection control. This study investigates the interplay between human neutrophils and T. gondii, exploring dynamics, key molecules, and signaling pathways involved in NETs production upon protozoan challenge. Methods and Results: Using confocal and electron microscopy, live cell imaging, pharmacological inhibitors, and DNA quantification assays, we find that human neutrophils promptly release both classical and rapid NETs upon pathogen stimulation. The NETs structure exhibits diverse phenotypes over time and is consistently associated with microorganisms. Mechanisms involve neutrophil elastase and peptidylarginine deiminase, along with intracellular calcium signaling and the PI3K pathway. Unexpectedly, human traps do not diminish viability or infectivity, but potentially aid in capturing parasites for subsequent neutrophil phagocytosis and elimination. Discussion: By revealing NETs formation mechanisms and their nuanced impact on T. gondii infection dynamics, our findings contribute to broader insights into host-pathogen relationships.

Silver(I) 1,10-Phenanthroline Complexes Are Active against Fonsecaea pedrosoi Viability and Negatively Modulate Its Potential Virulence Attributes.

The genus Fonsecaea is one of the etiological agents of chromoblastomycosis (CBM), a chronic subcutaneous disease that is difficult to treat. This work aimed to evaluate the effects of copper(II), manganese(II) and silver(I) complexes coordinated with 1,10-phenanthroline (phen)/1,10-phenanthroline-5,6-dione (phendione) on Fonsecaea spp. Our results revealed that most of these complexes were able to inhibit F. pedrosoi, F. monophora and F. nubica conidial viability with minimum inhibitory concentration (MIC) values ranging from 0.6 to 100 µM. The most effective complexes against F. pedrosoi planktonic conidial cells, the main etiologic agent of CBM, were [Ag(phen)2]ClO4 and [Ag2(3,6,9-tdda)(phen)4].EtOH, (tdda: 3,6,9-trioxaundecanedioate), displaying MIC values equal to 1.2 and 0.6 µM, respectively. These complexes were effective in reducing the viability of F. pedrosoi biofilm formation and maturation. Silver(I)-tdda-phen, combined with itraconazole, reduced the viability and extracellular matrix during F. pedrosoi biofilm development. Moreover, both silver(I) complexes inhibited either metallo- or aspartic-type peptidase activities of F. pedrosoi as well as its conidia into mycelia transformation and melanin production. In addition, the complexes induced the production of intracellular reactive oxygen species in F. pedrosoi. Taken together, our data corroborate the antifungal action of metal-phen complexes, showing they represent a therapeutic option for fungal infections, including CBM.

Chalcone Derivative Induces Flagellar Disruption and Autophagic Phenotype in Phytomonas serpens In Vitro.

Phytomonas serpens is a trypanosomatid phytoparasite, found in a great variety of species, including tomato plants. It is a significant problem for agriculture, causing high economic loss. In order to reduce the vegetal infections, different strategies have been used. The biological activity of molecules obtained from natural sources has been widely investigated to treat trypanosomatids infections. Among these compounds, chalcones have been shown to have anti-parasitic and anti-inflammatory effects, being described as having a remarkable activity on trypanosomatids, especially in Leishmania species. Here, we evaluated the antiprotozoal activity of the chalcone derivative (NaF) on P. serpens promastigotes, while also assessing its mechanism of action. The results showed that treatment with the derivative NaF for 24 h promotes an important reduction in the parasite proliferation (IC50/24 h = 23.6 ± 4.6 µM). At IC50/24 h concentration, the compound induced an increase in reactive oxygen species (ROS) production and a shortening of the unique flagellum of the parasites. Electron microscopy evaluation reinforced the flagellar phenotype in treated promastigotes, and a dilated flagellar pocket was frequently observed. The treatment also promoted a prominent autophagic phenotype. An increased number of autophagosomes were detected, presenting different levels of cargo degradation, endoplasmic reticulum profiles surrounding different cellular structures, and the presence of concentric membranar structures inside the mitochondrion. Chalcone derivatives may present an opportunity to develop a treatment for the P. serpens infection, as they are easy to synthesize and are low in cost. In order to develop a new product, further studies are still necessary.

Establishment of a murine model of congenital toxoplasmosis and validation of a qPCR assay to assess the parasite load in maternal and fetal tissues.

Toxoplasma gondii is the causative agent of toxoplasmosis, a disease that affects warm-blooded animals and one third of the human population worldwide. Pregnant women who have never been exposed to the parasite constitute an important risk group, as infection during pregnancy often leads to congenital toxoplasmosis, the most severe form of the disease. Current therapy for toxoplasmosis is the same as it was 50 years ago and has little or no effect when vertical transmission occurs. Therefore, it is urgent to develop new strategies to prevent mother-to-fetus transmission. The implementation of experimental animal models of congenital toxoplasmosis that reproduces the transmission rates and clinical signs in humans opens an avenue of possibilities to interfere in the progression of the disease. In addition, knowing the parasite load in maternal and fetal tissues after infection, which may be related to organ abnormalities and disease outcome, is another important step in designing a promising intervention strategy. Therefore, we implemented here a murine model of congenital toxoplasmosis with outbred Swiss Webster mice infected intravenously with tachyzoites of the ME49 strain of T. gondii that mimics the frequency of transmission of the parasite, as well as important clinical signs of human congenital toxoplasmosis, such as macrocephaly, in addition to providing a highly sensitive quantitative real-time PCR assay to assess parasite load in mouse tissues. As the disease is not restricted to humans, also affecting several domestic animals, including companion animals and livestock, they can also benefit from the model presented in this study.

Interleukin-27 Promotes Divergent Effects on HIV-1 Infection in Peripheral Blood Mononuclear Cells through BST-2/Tetherin.

Interleukin-27 (IL-27) is able to inhibit HIV-1 replication in peripheral blood mononuclear cells (PBMCs), macrophages, and dendritic cells. Here, we identify that IL-27 can produce opposing effects on HIV-1 replication in PBMCs and that the HIV-1 restriction factor BST-2/Tetherin is involved in both inhibitory and enhancing effects on HIV-1 infection induced by IL-27. IL-27 inhibited HIV-1 replication when added to cells 2 h after infection, promoting the prototypical BST-2/Tetherin-induced virion accumulation at the cell membrane of HIV-1-infected PBMCs. BST-2/Tetherin gene expression was significantly upregulated in the IL-27-treated PBMCs, with a simultaneous increase in the number of BST-2/Tetherin+ cells. The silencing of BST-2/Tetherin diminished the anti-HIV-1 effect of IL-27. In contrast, IL-27 increased HIV-1 production when added to infected cells 4 days after infection. This enhancing effect was prevented by BST-2/Tetherin gene knockdown, which also permitted IL-27 to function again as an HIV-1 inhibitory factor. These contrasting roles of IL-27 were associated with the dynamic of viral production, since the IL-27-mediated enhancement of virus replication was prevented by antiretroviral treatment of infected cells, as well as by keeping cells under agitation to avoid cell-to-cell contact. Likewise, inhibition of CD11a, an integrin associated with HIV-1 cell-to-cell transmission, abrogated the IL-27 enhancement of HIV-1 production. Our findings illustrate the complexity of the HIV-1-host interactions and may impact the potential therapeutic use of IL-27 and other soluble mediators that induce BST-2/Tetherin expression for HIV-1 infection. IMPORTANCE Here, we describe new findings related to the ability of the cytokine IL-27 to regulate the growth of HIV-1 in CD4+ T lymphocytes. IL-27 has long been considered a potent inhibitor of HIV-1 replication, a notion based on several reports showing that this cytokine controls HIV-1 infection in peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages, and dendritic cells. However, our present results are contrary to the current knowledge that IL-27 acts only as a powerful downregulator of HIV-1 replication. We observed that IL-27 can either prevent or enhance viral growth in PBMCs, an outcome dependent on when this cytokine is added to the infected cells. We detected that the increase of HIV-1 dissemination is due to enhanced cell-to-cell transmission with the involvement of the interferon-induced HIV-1 restriction factor BST-2/Tetherin and CD11a (LFA-1), an integrin that participates in formation of virological synapse.

Different Drugs, Same End: Ultrastructural Hallmarks of Autophagy in Pathogenic Protozoa.

Protozoan parasites interact with a wide variety of organisms ranging from bacteria to humans, representing one of the most common causes of parasitic diseases and an important public health problem affecting hundreds of millions of people worldwide. The current treatment for these parasitic diseases remains unsatisfactory and, in some cases, very limited. Treatment limitations together with the increased resistance of the pathogens represent a challenge for the improvement of the patient's quality of life. The continuous search for alternative preclinical drugs is mandatory, but the mechanisms of action of several of these compounds have not been described. Electron microscopy is a powerful tool for the identification of drug targets in almost all cellular models. Interestingly, ultrastructural analysis showed that several classes of antiparasitic compounds induced similar autophagic phenotypes in trypanosomatids, trichomonadids, and apicomplexan parasites as well as in Giardia intestinalis and Entamoeba spp. with the presence of an increased number of autophagosomes as well as remarkable endoplasmic reticulum profiles surrounding different organelles. Autophagy is a physiological process of eukaryotes that maintains homeostasis by the self-digestion of nonfunctional organelles and/or macromolecules, limiting redundant and damaged cellular components. Here, we focus on protozoan autophagy to subvert drug effects, discussing its importance for successful chemotherapy.

Physical Exercise Promotes a Reduction in Cardiac Fibrosis in the Chronic Indeterminate Form of Experimental Chagas Disease.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a neglected tropical disease and a health problem in Latin America. Etiological treatment has limited effectiveness in chronic CD; thus, new therapeutic strategies are required. The practice of physical exercises has been widely advocated to improve the quality of life of CD patients. The most frequent clinical CD manifestation is the chronic indeterminate form (CIF), and the effect of physical exercises on disease progression remains unknown. Here, in a CIF model, we aimed to evaluate the effect of physical exercises on cardiac histological, parasitological, mitochondrial, and oxidative metabolism, electro and echocardiographic profiles, and immunological features. To establish a CIF model, BALB/c and C57BL/6 mice were infected with 100 and 500 trypomastigotes of the Y T. cruzi strain. At 120 days postinfection (dpi), all mouse groups showed normal PR and corrected QT intervals and QRS complexes. Compared to BALB/c mice, C57BL/6 mice showed a lower parasitemia peak, mortality rate, and less intense myocarditis. Thus, C57BL/6 mice infected with 500 parasites were used for subsequent analyses. At 120 dpi, a decrease in cardiac mitochondrial oxygen consumption and an increase in reactive oxygen species (ROS) were detected. When we increased the number of analyzed mice, a reduced heart rate and slightly prolonged corrected QT intervals were detected, at 120 and 150 dpi, which were then normalized at 180 dpi, thus characterizing the CIF. Y-infected mice were subjected to an exercise program on a treadmill for 4 weeks (from 150 to 180 dpi), five times per week in a 30-60-min daily training session. At 180 dpi, no alterations were detected in cardiac mitochondrial and oxidative metabolism, which were not affected by physical exercises, although ROS production increased. At 120 and 180 dpi, comparing infected and non-infected mice, no differences were observed in the levels of plasma cytokines, indicating that a crucial biomarker of the systemic inflammatory profile was absent and not affected by exercise. Compared with sedentary mice, trained Y-infected mice showed similar parasite loads and inflammatory cells but reduced cardiac fibrosis. Therefore, our data show that physical exercises promote beneficial changes that may prevent CD progression.

Aedes aegypti Infection With Trypanosomatid Strigomonas culicis Alters Midgut Redox Metabolism and Reduces Mosquito Reproductive Fitness.

Aedes aegypti mosquitoes transmit arboviruses of important global health impact, and their intestinal microbiota can influence vector competence by stimulating the innate immune system. Midgut epithelial cells also produce toxic reactive oxygen species (ROS) by dual oxidases (DUOXs) that are essential players in insect immunity. Strigomonas culicis is a monoxenous trypanosomatid that naturally inhabits mosquitoes; it hosts an endosymbiotic bacterium that completes essential biosynthetic pathways of the parasite and influences its oxidative metabolism. Our group previously showed that S. culicis hydrogen peroxide (H2O2)-resistant (WTR) strain is more infectious to A. aegypti mosquitoes than the wild-type (WT) strain. Here, we investigated the influence of both strains on the midgut oxidative environment and the effect of infection on mosquito fitness and immunity. WT stimulated the production of superoxide by mitochondrial metabolism of midgut epithelial cells after 4 days post-infection, while WTR exacerbated H2O2 production mediated by increased DUOX activity and impairment of antioxidant system. The infection with both strains also disrupted the fecundity and fertility of the females, with a greater impact on reproductive fitness of WTR-infected mosquitoes. The presence of these parasites induced specific transcriptional modulation of immune-related genes, such as attacin and defensin A during WTR infection (11.8- and 6.4-fold, respectively) and defensin C in WT infection (7.1-fold). Thus, we propose that A. aegypti oxidative response starts in early infection time and does not affect the survival of the H2O2-resistant strain, which has a more efficient antioxidant system. Our data provide new biological aspects of A. aegypti-S. culicis relationship that can be used later in alternative vector control strategies.

In vitro effects of bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 on the physiology and interaction process of Leishmania amazonensis.

Leishmaniasis is one of the most relevant neglected tropical diseases in the world, affecting 14 million people. Despite the high morbidity, mortality and socio-economic impact, few therapeutic options are available for this disease. To make matters worse, the available molecules have several limitations such as limited efficacy, high cost, side effects and increased resistance. In this context, our group previously synthesized new compounds with anti-leishmania potential being the bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 (complex 4) the most promising one. Therefore, this present work revealed some morphological and physiological changes promoted by complex 4 on Leishmania amazonensis promastigotes as well as it was evidenced its potential against intramacrophage amastigotes. Complex 4 promoted a progressive reduction on the promastigotes size and a remarkable increase on the granularity/complexity as judged by flow cytometry. Transmission electron microscopy (TEM) analysis revealed extensive mitochondrial and plasma membrane alterations, although plasma membrane integrity remained. The mitochondrial changes observed by TEM were accompanied by a decrease in the activity of mitochondrial dehydrogenases with increased production of reactive oxygen species. Interestingly, promastigotes also showed changes in lipid metabolism. Besides the very low toxicity to macrophages, complex 4 had a great effect on intramacrophage amastigotes, displaying an IC50 of 3.91 µM. Collectively, the data presented here reinforce the potential of aminopyridyl compounds complexed to zinc against L. amazonensis. Thus, our work serves as a basis for in vivo assays to be designed or even the synthesis of more selective/effective compounds with lower cost.

Ruthenium(II)- and Palladium(II)-catalyzed position-divergent CH oxygenations of arylated quinones: Identification of hydroxylated quinonoid compounds with potent trypanocidal activity.

A diversity-oriented synthesis of hydroxylated aryl-quinones via CH oxygenation reactions and their evaluation against Trypanosoma cruzi, the etiological agent of Chagas disease, was accomplished. With the use of ruthenium(II)- or palladium(II)-based catalysts, complementary regioselectivities were observed in the hydroxylation reactions and we have identified 9 compounds more potent than benznidazole (Bz) among these novel arylated and hydroxylated quinones. For instance, 5-hydroxy-2-[4-(trifluoromethyl)phenyl]-1,4-naphthoquinone (4h) with an IC50/24 h value of 22.8 µM is 4.5-fold more active than the state-of-the-art drug Bz. This article provides the first example of the application of CH activation for the position-selective hydroxylation of arylated quinones and the identification of these compounds as trypanocidal drug candidates.

Novel N,N-di-alkylnaphthoimidazolium derivative of ß-lapachone impaired Trypanosoma cruzi mitochondrial electron transport system.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a neglected tropical disease that is endemic in Latin America and spreading worldwide due to globalization. The current treatments are based on benznidazole and nifurtimox; however, these drugs have important limitations and limited efficacy during the chronic phase, reinforcing the necessity of an alternative chemotherapy. For the last 30 years, our group has been evaluating the biological activity of naphthoquinones and derivatives on T. cruzi, and of the compounds tested, N1, N2 and N3 were found to be the most active in vitro. Here, we show the synthesis of a novel ß-lapachone-derived naphthoimidazolium named N4 and assess its activity on T. cruzi stages and the mechanism of action. The new compound was very active on all parasite stages (IC50/24 h in the range of 0.8-7.9 µM) and had a selectivity index of 5.4. Mechanistic analyses reveal that mitochondrial ROS production begins after short treatment starts and primarily affects the activity of complexes II-III. After 24 h treatment, a partial restoration of mitochondrial physiology (normal complexes II-III and IV activities and controlled H2O2 release) was observed; however, an extensive injury in its morphology was still detected. During treatment with N4, we also observed that trypanothione reductase activity increased in a time-dependent manner and concomitant with increased oxidative stress. Molecular docking calculations indicated the ubiquinone binding site of succinate dehydrogenase as an important interaction point with N4, as with the FMN binding site of dihydroorotate dehydrogenase. The results presented here may be a good starting point for the development of alternative treatments for Chagas disease and for understanding the mechanism of naphthoimidazoles in T. cruzi.

Naphthoquinones and Derivatives for Chemotherapy: Perspectives and Limitations of their Anti-trypanosomatids Activities.

Chagas disease, Sleeping sickness and Leishmaniasis, caused by trypanosomatids Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively, are considered neglected tropical diseases, and they especially affect impoverished populations in the developing world. The available chemotherapies are very limited, and a search for alternatives is still necessary. In folk medicine, natural naphthoquinones have been employed for the treatment of a great variety of illnesses, including parasitic infections. This review is focused on the anti-trypanosomatid activity and mechanistic analysis of naphthoquinones and derivatives. Among all the series of derivatives tested in vitro, naphthoquinone-derived 1,2,3-triazoles were very active on T. cruzi infective forms in blood bank conditions, as well as in amastigotes of Leishmania spp. naphthoquinones containing a CF3 on a phenyl amine ring inhibited T. brucei proliferation in the nanomolar range, and naphthopterocarpanquinones stood out for their activity on a range of Leishmania species. Some of these compounds showed a promising selectivity index (SI) (30 to 1900), supporting further analysis in animal models. Indeed, high toxicity to the host and inactivation by blood components are crucial obstacles to be overcome to use naphthoquinones and/or their derivatives for chemotherapy. Multidisciplinary initiatives embracing medicinal chemistry, bioinformatics, biochemistry, and molecular and cellular biology need to be encouraged to allow the optimization of these compounds. Large scale automated tests are pivotal for the efficiency of the screening step, and subsequent evaluation of both the mechanism of action in vitro and pharmacokinetics in vivo is essential for the development of a novel, specific and safe derivative, minimizing adverse effects.

Starvation and pH stress conditions induced mitochondrial dysfunction, ROS production and autophagy in Trypanosoma cruzi epimastigotes.

Chagas disease is a neglected illness endemic in Latin America that mainly affects rural populations. The etiological agent of Chagas disease is the protozoan Trypanosoma cruzi, which has three different parasite stages and a dixenous life cycle that includes colonization of the vertebrate and invertebrate hosts. During its life cycle, T. cruzi is subjected to stress conditions, including variations in nutrient availability and pH, which impact parasite survival and differentiation. The plasticity of mitochondrial function in trypanosomatids has been defined as mitochondrial activity related to substrate availability. Thus, mitochondrial remodeling and autophagy, which is a constitutive cellular process of turnover and recycling of cellular components, may constitute a response to the nutritional and pH stress in the host. To assess these processes, epimastigotes were subjected to acidic, alkaline, and nutritional stress conditions, and mitochondrial function and its influence on the autophagic process were evaluated. Our data demonstrated that the three stress conditions affected the mitochondrial structure, inducing organelle swelling and impaired oxidative phosphorylation. Stressed epimastigotes produced increased ROS levels and overexpressed antioxidant enzymes. The stress conditions resulted in an increase in the number of autophagosomes and exacerbated the expression of different autophagy-related genes (Atgs). A correlation between mitochondrial dysfunction and autophagic phenotypes was also observed. After 24 h, acid stress and nutritional deprivation induced metacyclogenesis phenotypes (mitochondrial remodeling and autophagy). On the other hand, alkaline stress was transient due to insect blood feeding and culminated in an increase in autophagic flux as a survival mechanism.

Chewing Lice of Fregata magnificens with First Record of Fregatiella aurifasciata (Phthiraptera: Amblycera) in Brazil.

The genus Fregata includes 5 species, with 3 recorded in Brazil, with Fregata magnificens being the most abundant. However, its ectoparasitic fauna is still little known. This study aimed to evaluate the incidence of ectoparasites of F. magnificens residing along the coast of Rio de Janeiro and São Paulo collected by 2 animal rehabilitation centers. Samples were collected from 5 frigatebirds of the Instituto Argonauta in São Paulo and 10 frigatebirds of the Centro de Recuperação de Animais Selvagens (CRAS) in Rio de Janeiro. Species of lice were identified using both morphological and molecular methods. Scanning electron microscopy was also used for identification. Colpocephalum spineum, Fregatiella aurifasciata, and Pectinopygus fregatiphagus were identified. All 3 louse species have previously been recorded from this host outside Brazil, but only P. fregatiphagus has been recorded from Brazil. This paper reports the first occurrence of F. aurifasciata and C. spineum in Brazil. It is also the first record of P. fregatiphagus in the state of Rio de Janeiro.

Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes.

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.

Strategies towards potent trypanocidal drugs: Application of Rh-catalyzed [2 + 2 + 2] cycloadditions, sulfonyl phthalide annulation and nitroalkene reactions for the synthesis of substituted quinones and their evaluation against Trypanosoma cruzi.

Rhodium-catalyzed [2 + 2 + 2] cycloadditions, sulfonyl phthalide annulations and nitroalkene reactions have been employed for the synthesis of 56 quinone-based compounds. These were evaluated against Trypanosoma cruzi, the parasite that causes Chagas disease. The reactions described here are part of a program that aims to utilize modern, versatile and efficient synthetic methods for the one or two step preparation of trypanocidal compounds. We have identified 9 compounds with potent activity against the parasite; 3 of these were 30-fold more potent than benznidazole (Bz), a drug used for the treatment of Chagas disease. This article provides a comprehensive outline of reactions involving over 120 compounds aimed at the discovery of new quinone-based frameworks with activity against T. cruzi.

The induction of host cell autophagy triggers defense mechanisms against Trypanosoma cruzi infection in vitro.

Trypanosoma cruzi causes Chagas disease, a neglected illness that affects millions of people worldwide, especially in Latin America. The balance between biochemical pathways triggered by the parasite and host cells response will ultimately define the progression of a life-threatening disease, justifying the efforts to understand cellular mechanisms for infection restrain. In this interaction, parasite and host cells are affected by different physiological responses as autophagy modulation, which could be under intense cellular stress, such as nutrient deprivation, hormone depletion, or infection. Autophagy is a constitutive pathway that leads to degradation of macromolecules and cellular structures and may induce cell death. In Trypanosoma cruzi infection, the relevance of host autophagy is controversial regarding in vitro parasite intracellular life cycle. In the present study, we evaluated host cell autophagy during T. cruzi infection in phagocytic and non-professional phagocytic cells. We described that the presence of the parasite increased the number of LC3 puncta, a marker for autophagy, in cardiac cells and peritoneal macrophages in vitro. The induction of host autophagy decreased infection in macrophages in early and late time-periods. We suggest that starved phagocytic cells reduced internalization, also confirmed by inert particles and dead trypomastigotes. Whereas, in cardiac cells, starvation-induced autophagy decreased lipid droplets and infection in later time-point, by reducing parasite differentiation/proliferation. In ATG5 knockout MEF cells, we confirmed our hypothesis of autophagy machinery activation during parasite internalization, increasing infection. Our data suggest that host autophagy downregulates T. cruzi infection through impairing parasite intracellular life cycle, reducing the infection in primary culture cells.

Rapamycin Treatment Reduces Acute Myocarditis Induced by Trypanosoma cruzi Infection.

Chagas disease affects millions of people mainly in Latin America and is a protozoan illness caused by the parasite Trypanosoma cruzi. Chagasic cardiomyopathy is the leading cause of mortality of infected patients, due to compromised electrical and mechanical cardiac function induced by tissue remodeling, especially fibrosis, and lymphocytic infiltration. Some cellular biochemical pathways can be protective to the heart, and we tested if the in vivo activation of the autophagic machinery by rapamycin could reduce parasite-induced myocarditis. Regarding the expression of LC3, an autophagy marker, we observed its upregulation in the cardiac tissue of infected untreated mice. However, after rapamycin treatment, an autophagy inducer, infected mice showed reduced electrical cardiac dysfunctions, myocarditis, cardiac damage, and reduced production of pro-inflammatory cytokines by the heart. On the other hand, the parasite's life cycle was not affected, and we observed no modulations in cardiac tissue or blood parasitemia. Our data indicate that, at least partially, autophagy induction controls inflammation in the heart¸ illustrating the complexity of the pathways that concur to the development of the infection.

Quantitative Proteomic Map of the Trypanosomatid Strigomonas culicis: The Biological Contribution of its Endosymbiotic Bacterium.

Strigomonas culicis is a kinetoplastid parasite of insects that maintains a mutualistic association with an intracellular symbiotic bacterium, which is highly integrated into the protist metabolism: it furnishes essential compounds and divides in synchrony with the eukaryotic nucleus. The protist, conversely, can be cured of the endosymbiont, producing an aposymbiotic cell line, which presents a diminished ability to colonize the insect host. This obligatory association can represent an intermediate step of the evolution towards the formation of an organelle, therefore representing an interesting model to understand the symbiogenesis theory. Here, we used shotgun proteomics to compare the S. culicis endosymbiont-containing and aposymbiotic strains, revealing a total of 11,305 peptides, and up to 2,213 proteins (2,029 and 1,452 for wild type and aposymbiotic, respectively). Gene ontology associated to comparative analysis between both strains revealed that the biological processes most affected by the elimination of the symbiont were the amino acid synthesis, as well as protein synthesis and folding. This large-scale comparison of the protein expression in S. culicis marks a step forward in the comprehension of the role of endosymbiotic bacteria in monoxenous trypanosomatid biology, particularly because trypanosomatids expression is mostly post-transcriptionally regulated.