Extracellular Vesicles from Scedosporium apiospermum Mycelial Cells: Implication for Fungal-Host Interplays.

The release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs' protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.

Manganese(II), copper(II) and silver(I) complexes containing 1,10-phenanthroline/1,10-phenanthroline-5,6-dione against Candida species.

Background: New chemotherapeutics are urgently required to treat Candida infections caused by drug-resistant strains. Methods: The effects of 16 1,10-phenanthroline (phen)/1,10-phenanthroline-5,6-dione/dicarboxylate complexed with Mn(II), Cu(II) and Ag(I) were evaluated against ten different Candida species. Results: Proliferation of Candida albicans, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis and Candida tropicalis was inhibited by three of six Cu(II) (MICs 1.52-21.55 µM), three of three Ag(I) (MICs 0.11-12.74 µM) and seven of seven Mn(II) (MICs 0.40-38.06 µM) complexes. Among these [Mn2(oda)(phen)4(H2O)2][Mn2(oda)(phen)4(oda)2].4H2O, where oda = octanedioic acid, exhibited effective growth inhibition (MICs 0.4-3.25 µM), favorable activity indexes, low toxicity against Vero cells and good/excellent selectivity indexes (46.88-375). Conclusion: [Mn2(oda)(phen)4(H2O)2][Mn2(oda)(phen)4(oda)2].4H2O represents a promising chemotherapeutic option for emerging, medically relevant and drug-resistant Candida species.

Candida species are widespread fungi that can cause a variety of infections in humans, and some of them exhibit resistance profile to existing antifungal drugs. Consequently, it is imperative to discover novel treatments for these clinically relevant human infections. Complexes are chemical compounds containing metal ion components that are well-known for their antimicrobial properties, including antifungal activity. In the present study, we investigated the effects of 16 novel complexes against ten medically relevant Candida species, including some strains resistant to commonly used clinical antifungals. Our findings revealed that all complexes containing manganese and silver metals effectively inhibited the growth of all Candida species tested, albeit to varying extents. Some of these complexes exhibited superior antifungal activity and lower toxicity to mammalian cells compared to traditional antifungals, such as fluconazole. In conclusion, these new complexes hold promise as a potential novel approach for treating fungal infections, especially those caused by drug-resistant Candida strains.

Chagas Disease Control-Many Approaches to Prospect.

Chagas disease is an emerging and neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, estimated to infect 8 to 10 million people worldwide, according to the World Health Organization [...].

In Vitro Effects of Aminopyridyl Ligands Complexed to Copper(II) on the Physiology and Interaction Process of Trypanosoma cruzi.

Chagas disease is derived from the infection by the protozoan Trypanosoma cruzi. In many countries, benznidazole is the only drug approved for clinical use despite several side effects and the emergence of resistant parasite strains. In this context, our group has previously pointed out that two novel aminopyridine derivatives complexed with Cu2+, namely, cis-aquadichloro(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamino)copper (3a) and its glycosylated ligand cis-dichloro (N-{[4-(2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyloxy)pheny]lmethyl}-2-pyridinemethamino)copper (3b), are effective against T. cruzi trypomastigote forms. With this result in mind, the present work aimed to investigate the effects of both compounds on trypomastigotes physiology and on the interaction process with host cells. Apart from loss of plasma membrane integrity, an increased generation of reactive oxygen species (ROS) and decreased mitochondrial metabolism were observed. Pretreatment of trypomastigotes with these metallodrugs inhibited the association index with LLC-MK2 cells in a typical dose-dependent manner. Both compounds showed low toxicity on mammalian cells (CC50 > 100 µM), and the IC50 values calculated for intracellular amastigotes were determined as 14.4 µM for 3a and 27.1 µM for 3b. This set of results demonstrates the potential of these aminopyridines complexed with Cu2+ as promising candidates for further antitrypanosomal drug development.

Antileishmanial Efficacy of the Calpain Inhibitor MDL28170 in Combination with Amphotericin B.

The necessity of drug combinations to treat leishmaniasis came to the surface mainly because of the toxicity of current treatments and the emergence of resistant strains. The calpain inhibitor MDL28170 has previously shown anti-Leishmania activity, therefore its use in association with standard drugs could provide a new alternative for the treatment strategy against leishmaniasis. In this study, we analyzed the potential of the combination of MDL28170 and the antileishmanial drug amphotericin B against Leishmania amazonensis and Leishmania chagasi. The compounds were tested in the combination of the ½ × IC50 value of MDL28170 plus the » × IC50 value of amphotericin B, which led to an increment in the anti-promastigote activity when compared to the single drug treatments. This drug association revealed several and severe morphophysiological changes on parasite cells, such as loss of plasma membrane integrity, reduced size of flagellum, and depolarization of mitochondrial membrane potential besides increased reactive oxygen species production. In addition, the combination of both drugs had a deleterious effect on the Leishmania-macrophage interaction, reflecting in a significant anti-amastigote action, which achieved a reduction of 50% in the association index. These results indicate that the combination treatment proposed here may represent a new alternative for leishmaniasis chemotherapy.

Lopinavir and Nelfinavir Induce the Accumulation of Crystalloid Lipid Inclusions within the Reservosomes of Trypanosoma cruzi and Inhibit Both Aspartyl-Type Peptidase and Cruzipain Activities Detected in These Crucial Organelles.

Several research groups have explored the repositioning of human immunodeficiency virus aspartyl peptidase inhibitors (HIV-PIs) on opportunistic infections caused by bacteria, fungi and protozoa. In Trypanosoma cruzi, HIV-PIs have a high impact on parasite viability, and one of the main alterations promoted by this treatment is the imbalance in the parasite's lipid metabolism. However, the reasons behind this phenomenon are unknown. In the present work, we observed by transmission electron microscopy (TEM) that the treatment of T. cruzi epimastigotes with the HIV-PIs lopinavir and nelfinavir induced a huge accumulation of crystalloid-shaped lipids within the reservosomes, most of them deforming these key organelles. As previously reported, those structures are characteristic of lipid inclusions formed mostly of cholesterol and cholesterol-esters. The fractionation of nontreated epimastigotes generated two distinct fractions enriched in reservosomes: one mostly composed of lipid inclusion-containing reservosomes (Fraction B1) and one where lipid inclusions were much less abundant (Fraction B2). Interestingly, the extract of Fraction B2 presented enzymatic activity related to aspartyl-type peptidases 3.5 times higher than that found in the extract obtained from Fraction B1. The cleavage of cathepsin D substrate by this class of peptidases was strongly impaired by pepstatin A, a prototypical aspartyl PI, and the HIV-PIs lopinavir and nelfinavir. In addition, both HIV-PIs also inhibited (to a lesser extent) the cruzipain activity present in reservosomes. Finally, our work provides new evidence concerning the presence and supposed participation of aspartyl peptidases in T. cruzi, even as it adds new information about the mechanisms behind the alterations promoted by lopinavir and nelfinavir in the protozoan.

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.

Naringenin-Functionalized Multi-Walled Carbon Nanotubes: A Potential Approach for Site-Specific Remote-Controlled Anticancer Delivery for the Treatment of Lung Cancer Cells.

Multi-walled carbon nanotubes functionalized with naringenin have been developed as new drug carriers to improve the performance of lung cancer treatment. The nanocarrier was characterized by Transmission Electron Microscopy (TEM), Fourier-Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy, Raman Spectroscopy, and Differential Scanning Calorimetry (DSC). Drug release rates were determined in vitro by the dialysis method. The cytotoxic profile was evaluated using the MTT assay, against a human skin cell line (hFB) as a model for normal cells, and against an adenocarcinomic human alveolar basal epithelial (A569) cell line as a lung cancer in vitro model. The results demonstrated that the functionalization of carbon nanotubes with naringenin occurred by non-covalent interactions. The release profiles demonstrated a pH-responsive behavior, showing a prolonged release in the tumor pH environment. The naringenin-functionalized carbon nanotubes showed lower cytotoxicity on non-malignant cells (hFB) than free naringenin, with an improved anticancer effect on malignant lung cells (A549) as an in vitro model of lung cancer.

Funding for Chagas Disease: A 10-Year (2009-2018) Survey.

Chagas disease was discovered in 1909 by the Brazilian scientist Carlos Chagas. After more than 110 years, many outcomes have been achieved in all research fields; however, Chagas disease remains a serious public health problem, mainly in Latin America, being one of the most neglected tropical diseases in the world. As a neglected disease, it receives very little financial support. Nevertheless, how much is actually spent? With this question in mind, the goal of the present work was to summarize all funding employed by multiple institutions in the Chagas disease field in a 10-year survey. From 2009 to 2018, Chagas disease received only USD 236.31 million, representing 0.67% of the total applied for all neglected diseases in this period. Mostly, the investments are concentrated in basic research (47%) and drug development (42.5%), with the public sector responsible for 74% of all funding, followed by the industry (19%) and philanthropy (7%). Relevantly, NIH (USA) alone accounted for more than half of the total investment. Taking into account that Chagas disease has a great socio-economic impact, it is clear that more investments are needed, especially from endemic countries. Furthermore, coordinated strategies to make better use of resources and incentives for the pharmaceutical industry must be adopted.

In vitro effects of the asymmetric peptidomimetic 157, containing l-tartaric acid core and valine/leucine substituents, on Leishmania amazonensis promastigotes and amastigotes.

The current treatments for leishmaniasis bump into several obstacles, including low efficacy, high costs, long monitoring, and several/severe side effects. Consequently, the search for promising compounds is a tangible need. Recently, we reported the anti-Leishmania amazonensis action of asymmetric peptidomimetic compounds containing tartaric acid as core, especially the 157 derivative that contains valine/leucine substituents in its structure. Herein, we decipher the multiple effects of 157 on the L. amazonensis physiology and on the interaction process with macrophages. The peptidomimetic 157 induced significant changes on the morphometric (internal granularity reduction as judged by flow cytometer) and on the ultrastructural (round-shaped parasites, presence of plasma membrane blebs and flagellum loss as visualized by scanning electron microscopy) aspects of treated promastigotes compared to untreated ones. The alteration on the plasma membrane permeability was confirmed by the passive incorporation of propidium iodide in 157-treated promastigotes. In parallel, the low viability of promastigotes was also associated to the perturbation of mitochondrial transmembrane electric potential. These combined results demonstrated that 157 induced irreversible metabolic damages that led to L. amazonensis death. The pre-treatment of promastigotes with 157 inhibited the association index with macrophages in a typically dose-dependent manner. Additionally, 157 significantly reduced the number of intramacrophage amastigotes after 72 h of drug contact, presenting an IC50 value of 30.2 µM. Under our experimental conditions, 157 showed higher toxicity to promastigotes and amastigotes when compared to RAW cells, resulting in good selective indexes. Therefore, 157 can be considered as an interesting candidate for further optimization, since its synthesis is simple and cheap.

Repositioning of HIV Aspartyl Peptidase Inhibitors for Combating the Neglected Human Pathogen Trypanosoma cruzi.

Chagas disease, caused by the flagellate parasite Trypanosoma cruzi, is a wellknown neglected tropical disease. This parasitic illness affects 6-7 million people and can lead to severe myocarditis and/or complications of the digestive tract. The changes in its epidemiology facilitate co-infection with the Human Immunodeficiency Virus (HIV), making even more difficult the diagnosis and prognosis. The parasitic infection is reactivated in T. cruzi/HIV co-infection, with the appearance of unusual manifestations in the chronic phase and the exacerbation of classical clinical signs. The therapeutic arsenal to treat Chagas disease, in all its clinical forms, is restricted basically to two drugs, benznidazole and nifurtimox. Both drugs are extremely toxic and the therapeutic efficacy is still unclear, making the clinical treatment a huge issue to be solved. Therefore, it seems obvious the necessity of new tangible approaches to combat this illness. In this sense, the repositioning of approved drugs appears as an interesting and viable strategy. The discovery of Human Immunodeficiency Virus Aspartyl Peptidase Inhibitors (HIV-PIs) represented a milestone in the treatment of Acquired Immune Deficiency Syndrome (AIDS) and, concomitantly, a marked reduction in both the incidence and prevalence of important bacterial, fungal and parasitic co-infections was clearly observed. Taking all these findings into consideration, the present review summarizes the promising and beneficial data concerning the effects of HIV-PIs on all the evolutionary forms of T. cruzi and in important steps of the parasite's life cycle, which highlight their possible application as alternative drugs to treat Chagas disease.

The serine peptidase inhibitor TPCK induces several morphophysiological changes in the opportunistic fungal pathogen Candida parapsilosis sensu stricto.

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.

Docking simulation between HIV peptidase inhibitors and Trypanosoma cruzi aspartyl peptidase.

OBJECTIVE: The low investment in research, diagnosis and treatment are factors that contribute to the continuity of Chagas' disease as a neglected tropical diseases (NTDs). In this context, the repositioning of drugs represents a useful strategy, in the search for new chemotherapeutic approaches for NTDs. HIV aspartic peptidase inhibitors (HIV IPs) are good candidates for drug repurposing. Here, we modeled the three dimensional structure of an aspartyl peptidase of Trypanosoma cruzi, the causative agent of Chagas' disease, aligned it to the HIV aspartyl peptidase and performed docking binding assays with the HIV PIs. RESULTS: The 3D structure confirmed the presence of acid aspartic residues, which are critical to enzyme activity. The docking experiment revealed that HIV IPs bind to the active site of the enzyme, being ritonavir and lopinavir the ones with greater affinity. Benznidazole presented the worst binding affinity, this drug is currently used in Chagas' disease treatment and was included as negative control. These results together with previous data on the trypanocidal effect of the HIV PIs support the hypothesis that a T. cruzi aspartyl peptidase can be the intracellular target of these inhibitors. However, the direct demonstration of the inhibition of T. cruzi aspartyl peptidase activity by HIV PIs is still a goal to be persuaded.

Ultrastructural viewpoints on the interaction events of Scedosporium apiospermum conidia with lung and macrophage cells.

BACKGROUND: Scedosporium apiospermum is a ubiquitous, emerging and multidrug-resistant fungal pathogen with still rather unknown virulence mechanisms. OBJECTIVES/METHODS: The cellular basis of the in vitro interaction between fungi and host cells/tissues is the determinant factor for the development of a successful in vivo infection. Herein, we evaluated the interaction of S. apiospermum conidia with lung epithelial (A549), lung fibroblast (MRC-5) and RAW 264.7 macrophages by light and scanning/transmission electron microscopy. FINDINGS: After 4 h of fungi-host cell contact, the percentage of infected mammalian cells and the number of fungi per infected cell was measured by light microscopy, and the following association indexes were calculated for A549, MRC-5 and macrophage cells: 73.2 ± 25.9, 69.7 ± 22.5 and 59.7 ± 11.1, respectively. Both conidia and germinated conidia were regularly observed interacting with the evaluated cells, with a higher prevalence of non-germinated conidia. Interestingly, nests of germinated conidia were evidenced at the surface of lung cells by scanning electron microscopy. Some germination projections and hyphae were seen penetrating/evading the mammalian cells. Furthermore, internalised conidia were seen within vacuoles as visualised by transmission electron microscopy. MAIN CONCLUSIONS: The present study contributes to a better understanding of S. apiospermum pathogenesis by demonstrating the first steps of the infection process of this opportunistic fungus.

Primary evidence of the mechanisms of action of HIV aspartyl peptidase inhibitors on Trypanosoma cruzi trypomastigote forms.

The development of HIV aspartyl peptidase inhibitors (HIV-PIs) and their introduction into AIDS therapy preceded a significant decrease in the incidence, morbidity and mortality of relevant protozoan co-infections. However, few data are available about how HIV-PIs act on pathogenic parasites, such as Trypanosoma cruzi, the etiological agent of Chagas disease. Therefore, the aim of the present work was to evaluate different physiological aspects of the treatment of the infective trypomastigote forms of T. cruzi with the HIV-PIs, nelfinavir and lopinavir. At the LD50/4 h doses, both HIV-PIs significantly reduced the trypomastigote size and markedly increased the granularity/complexity. Transmission electron microscopy analysis associated to biochemical assays permitted definition of the main HIV-PIs targets in the parasite. Lopinavir and nelfinavir induced (i) plasma membrane shedding, particularly in the flagellar region, which drastically affected parasite integrity; (ii) strong mitochondrial swelling with rare matrix fragmentation, which were linked to severely reduced hydrolytic activity of dehydrogenases and organelle membrane depolarization; (iii) increased generation of reactive oxygen species (ROS); (iv) dilation of both nuclear envelope (without DNA disruption) and endoplasmic reticulum (with formation of autophagosomes), and (v) accumulation of intracellular lipid droplets, revealing a typical lipid metabolism disorder. Collectively, our study demonstrated that nelfinavir and lopinavir target vital cellular structures of trypomastigotes, culminating in irreversible metabolic injuries that lead to T. cruzi death.

Asymmetric peptidomimetics containing L-tartaric acid core inhibit the aspartyl peptidase activity and growth of Leishmania amazonensis promastigotes.

Aspartyl-type peptidases are promising chemotherapeutic targets in protozoan parasites. In the present work, we identified an aspartyl peptidase activity from the soluble extract of Leishmania amazonensis promastigotes, which cleaved the fluorogenic peptide 7-methoxycoumarin-4-acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-D-Arg-amide (cathepsin D substrate) under acidic pH conditions at 37°C, showing a KM of 0.58 µM and Vmax of 129.87 fluorescence arbitrary units/s mg protein. The leishmanial aspartyl peptidase activity was blocked by pepstatin A (IC50 = 6.8 µM) and diazo-acetyl-norleucinemetilester (IC50 = 10.2 µM), two classical aspartyl peptidase inhibitors. Subsequently, the effects of 6 asymmetric peptidomimetics, containing L-tartaric acid core, were tested on both aspartyl peptidase and growth of L. amazonensis promastigotes. The peptidomimetics named 88, 154 and 158 promoted a reduction of 50% on the leishmanial aspartyl peptidase activity at concentrations ranging from 40 to 85 µM, whereas the peptidomimetic 157 was by far the most effective, presenting IC50 of 0.04 µM. Furthermore, the peptidomimetics 157 and 154 reduced the parasite proliferation in a dose-dependent manner, displaying IC50 values of 33.7 and 44.5 µM, respectively. Collectively, the peptidomimetic 157 was the most efficient compound able to arrest both aspartyl peptidase activity and leishmanial proliferation, which raises excellent perspectives regarding its use against this human pathogenic protozoan.

Ultrastructural viewpoints on the interaction events of Scedosporium apiospermum conidia with lung and macrophage cells

BACKGROUND Scedosporium apiospermum is a ubiquitous, emerging and multidrug-resistant fungal pathogen with still rather unknown virulence mechanisms. OBJECTIVES/METHODS The cellular basis of the in vitro interaction between fungi and host cells/tissues is the determinant factor for the development of a successful in vivo infection. Herein, we evaluated the interaction of S. apiospermum conidia with lung epithelial (A549), lung fibroblast (MRC-5) and RAW 264.7 macrophages by light and scanning/transmission electron microscopy. FINDINGS After 4 h of fungi-host cell contact, the percentage of infected mammalian cells and the number of fungi per infected cell was measured by light microscopy, and the following association indexes were calculated for A549, MRC-5 and macrophage cells: 73.2 ± 25.9, 69.7 ± 22.5 and 59.7 ± 11.1, respectively. Both conidia and germinated conidia were regularly observed interacting with the evaluated cells, with a higher prevalence of non-germinated conidia. Interestingly, nests of germinated conidia were evidenced at the surface of lung cells by scanning electron microscopy. Some germination projections and hyphae were seen penetrating/evading the mammalian cells. Furthermore, internalised conidia were seen within vacuoles as visualised by transmission electron microscopy. MAIN CONCLUSIONS The present study contributes to a better understanding of S. apiospermum pathogenesis by demonstrating the first steps of the infection process of this opportunistic fungus.

The potent cell permeable calpain inhibitor MDL28170 affects the interaction of Leishmania amazonensis with macrophages and shows anti-amastigote activity.

Since the discovery of the28 first drugs used in leishmaniasis treatment up to now, the search for compounds with anti-Leishmania activity without toxic effects and able to overcome the emergency of resistant strains remains a major goal to combat this neglected disease. With this in mind, in the present work, we evaluated the effects of the calpain inhibitor MDL28170 on the interaction process of Leishmania amazonensis promastigote forms with murine peritoneal macrophages and on the intracellular amastigotes. Our results showed that the calpain inhibitor MDL28170 at 15 and 30µM significantly reduced the interaction process of promastigotes with macrophages by 16% and 41%, respectively. The inhibitor was also able to drastically reduce the number of infected macrophages in a time- and dose-dependent manner: after only 24h, MDL28170 was able to significantly diminish the infection rate, presenting an IC50 value of 18.2µM for amastigotes. The treatment with MDL28170 did not alter the nitric oxide production, but the production of TNF-α was significantly raised. Altogether, the results presented here contribute to the search of new proteolytic inhibitors able to act in a selective and effective manner against the diseases caused by trypanosomatids.

The Widespread Anti-Protozoal Action of HIV Aspartic Peptidase Inhibitors: Focus on Plasmodium spp., Leishmania spp. and Trypanosoma cruzi.

The introduction of the HIV aspartic peptidase inhibitors (HIV-PIs) has revolutionized the medical arena, since they have drastically reduced the number and the severity of opportunistic infections, including the protozoal diseases that afflict the HIV-infected individuals worldwide. HIV-PIs rapidly and profoundly diminish the viral load, which is paralleled by increase in the CD4+ T lymphocyte counts and stimulation of the survival and activation of neutrophil, monocyte and endothelial cells, culminating in a vigorous reduction in the number of deaths due to the AIDS, in the number of new cases of AIDS and in the number of hospitalization days. Many research groups around the globe are trying to decipher both the in vitro and in vivo antiprotozoal mechanisms behind the use of HIVPIs. These studies have been supported by the urgent need to discover novel active compounds able to treat incurable parasitoses, including three major neglected diseases: malaria, leishmaniasis and Chagas' disease. The present review summarizes the recent advances on the effects of HIV-PIs against Plasmodium spp., Leishmania spp. and Trypanosoma cruzi.

Nelfinavir and lopinavir impair Trypanosoma cruzi trypomastigote infection in mammalian host cells and show anti-amastigote activity.

There is an urgent need to implement new strategies and to search for new chemotherapeutic targets to combat Chagas' disease. In this context, repositioning of clinically approved drugs appears as a viable tool to combat this and several other neglected pathologies. An example is the use of aspartic peptidase inhibitors (PIs) currently applied in human immunodeficiency virus (HIV) treatment against different infectious agents. Therefore, the main objective of this work was to verify the effects of the HIV-PIs nelfinavir and lopinavir against Trypanosoma cruzi using in vitro models of infection. Cytotoxicity assays with LLC-MK2 epithelial cells and RAW macrophages allowed an evaluation of the effects of HIV-PIs on the interaction between trypomastigotes and these cells as well as the survival of intracellular amastigotes. Pre-treatment of trypomastigotes with nelfinavir and lopinavir inhibited the association index with LLC-MK2 cells and RAW macrophages in a dose- and time-dependent manner. In addition, nelfinavir and lopinavir also significantly reduced the number of intracellular amastigotes in both mammalian cell lineages, particularly when administered in daily doses. Both compounds had no effect on nitric oxide production in infected RAW macrophages. These results open the possibility for the use of HIV-PIs as a tangible alternative in the treatment of Chagas' disease. However, the main mechanism of action of nelfinavir and lopinavir has yet to be elucidated, and more studies using in vivo models must be conducted.