Leishmaniases are neglected infectious diseases caused by parasites of the 'protozoan' genus
. Depending on the parasite species, different clinical forms are known as cutaneous, muco-cutaneous, and ...the visceral leishmaniasis (VL). VL is particularly fatal and the therapy presents limitations. In the search for new anti-leishmanial hit compounds, seven natural sesquiterpene lactones were evaluated against promastigotes and intracellular amastigotes of
(Leishmania)
, a pathogen causing VL. The pseudoguaianolides mexicanin I and helenalin acetate demonstrated the highest selectivity and potency against intracellular amastigotes. In addition, promastigotes treated with helenalin acetate were subject to an ultrastructural and biochemical investigation. The lethal action of the compound was investigated by fluorescence-activated cell sorting and related techniques to detect alterations in reactive oxygen species (ROS) content, plasma membrane permeability, and mitochondrial membrane potential. Helenalin acetate significantly reduced the mitochondrial membrane potential and the mitochondrial structural damage was also confirmed by transmission electron microscopy, displaying an intense organelle swelling. No alteration of plasma membrane permeability or ROS content could be detected. Additionally, helenalin acetate significantly increased the production of nitric oxide in peritoneal macrophages, probably potentiating the activity against the intracellular amastigotes. Helenalin acetate could hence be a useful anti-leishmanial scaffold for further optimization studies.
Therapeutic vaccines based on monocyte-derived dendritic cells have been shown to be promising strategies and may act as complementary treatments for viral infections, cancers, and, more recently, ...autoimmune diseases. Alpha-type-1-polarized dendritic cells (aDC1s) have been shown to induce type-1 immunity with a high capacity to produce interleukin-12p70 (IL-12p70). In the clinical use of cell-based therapeutics, injectable solutions can affect the morphology, immunophenotypic profile, and viability of cells before delivery and their survival after injection. In this sense, preparing a cell suspension that maintains the quality of aDC1s is essential to ensure effective immunotherapy. In the present study, monocytes were differentiated into aDC1s in the presence of IL-4 and GM-CSF. On day 5, the cells were matured by the addition of a cytokine cocktail consisting of IFN-α , IFN-γ , IL-1β , TNF-α , and Poly I:C. After 48hr, mature aDC1s were harvested and suspended in two different solutions: normal saline and Ringer's lactate. The maintenance of cells in suspension was evaluated after 4, 6, and 8hr of storage. Cell viability, immunophenotyping, and apoptosis analyses were performed by flow cytometry. Cellular morphology was observed by electron microscopy, and the production of IL-12p70 by aDC1s was evaluated by ELISA. Compared with normal saline, Ringer's lactate solution was more effective at maintaining DC viability for up to 8hr of incubation at 4 or 22°C.
The dioctadecyldimethylammonium bromide (DODAB) is a double-chained cationic lipid with potent bactericide and fungistatic activities; however, its toxicity on protozoan parasites is still unknown. ...Here, we show the antileishmanial activity of DODAB nano-sized cationic bilayer fragments on stationary-phase promastigotes and amastigotes of
, the causative agent of cutaneous leishmaniasis. Upon treatment with DODAB, we analyzed the parasite surface zeta-potential, parasite viability, cellular structural modifications, and intracellular proliferation. The DODAB cytotoxic effect was dose-dependent, with a median effective concentration (EC
) of 25 µM for both life-cycle stages, comparable to the reported data for bacteria and fungi. The treatment with DODAB changed the membrane zeta-potential from negative to positive, compromised the parasite's morphology, affected the cell size regulation, caused a loss of intracellular organelles, and probably dysregulated the plasma membrane permeability without membrane disruption. Moreover, the parasites that survived after treatment induced small parasitophorous vacuoles and failed to proliferate inside macrophages. In conclusion, DODAB displayed antileishmanial activity, and it remains to be elucidated how DODAB acts on the protozoan membrane. Understanding this mechanism can provide insights into the development of new parasite-control strategies.
Endothelial heterogeneity has important implications in health and disease. Molecular markers selectively expressed in the vasculature of different organs and tissues are currently being explored in ...targeted therapies with promising results in preclinical and clinical studies. Noteworthy is the role that combinatorial approaches such as phage display have had in identifying such markers by using phage as nanoparticles and surrogates for billions of different peptides, screening noninvasively the vascular lumen for binding sites. Here, we show that a new peptide motif that emerged from such combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine–Arginine–Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif.
Carajurin is the main constituent of
species with reported anti-
activity. However, its mechanism of action has not been described. This study investigated the mechanisms of action of carajurin ...against promastigote forms of
. Carajurin was effective against promastigotes with IC
of 7.96 ± 1.23 μg.mL
(26.4 µM), and the cytotoxic concentration for peritoneal macrophages was 258.2 ± 1.20 μg.mL
(856.9 µM) after 24 h of treatment. Ultrastructural evaluation highlighted pronounced swelling of the kinetoplast with loss of electron-density in
promastigotes induced by carajurin treatment. It was observed that carajurin leads to a decrease in the mitochondrial membrane potential (
= 0.0286), an increase in reactive oxygen species production (
= 0.0286), and cell death by late apoptosis (
= 0.0095) in parasites. Pretreatment with the antioxidant NAC prevented ROS production and significantly reduced carajurin-induced cell death. The electrochemical and density functional theory (DFT) data contributed to support the molecular mechanism of action of carajurin associated with the ROS generation, for which it is possible to observe a correlation between the LUMO energy and the electroactivity of carajurin in the presence of molecular oxygen. All these results suggest that carajurin targets the mitochondria in
. In addition, when assessed for its drug-likeness, carajurin follows Lipinski''s rule of five, and the Ghose, Veber, Egan, and Muegge criteria.
The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated ...for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis.
Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis-infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden.
The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.
Leishmaniasis is a neglected disease that affects more than 12 million people, with a limited therapy. Plant-derived natural products represent a useful source of anti-protozoan prototypes. In this ...work, four derivatives were prepared from neolignans isolated from the Brazilian plant Nectandra leucantha, and their effects against intracellular amastigotes of Leishmania (L.) infantum evaluated in vitro. IC
values between 6 and 35 µM were observed and in silico predictions suggested good oral bioavailability, no PAINS similarities, and ADMET risks typical of lipophilic compounds. The most selective (SI > 32) compound was chosen for lethal action and immunomodulatory studies. This compound caused a transient depolarization of the plasma membrane potential and induced an imbalance of intracellular Ca
, possibly resulting in a mitochondrial impairment and leading to a strong depolarization of the membrane potential and decrease of ATP levels. The derivative also interfered with the cell cycle of Leishmania, inducing a programmed cell death-like mechanism and affecting DNA replication. Further immunomodulatory studies demonstrated that the compound eliminates amastigotes via an independent activation of the host cell, with decrease levels of IL-10, TNF and MCP-1. Additionally, this derivative caused no hemolytic effects in murine erythrocytes and could be considered promising for future lead studies.
It has been estimated that individuals with COVID‐19 can shed replication‐competent virus up to a maximum of 20 days after initiation of symptoms. The majority of studies that addressed this ...situation involved hospitalized individuals and those with severe disease. Studies to address the possible presence of SARS‐CoV‐2 during the different phases of COVID‐19 disease in mildly infected individuals, and utilization of viral culture techniques to identify replication‐competent viruses, have been limited. This report describes two patients with mild forms of the disease who shed replication‐competent virus for 24 and 37 days, respectively, after symptom onset.
Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are cardioactive steroids ...from animals and plants that have also been reported to possess antimicrobial activities. Leishmaniasis and American Trypanosomiasis are parasitic diseases found in tropical and subtropical regions. The efforts toward the discovery of new treatments for these diseases have been largely neglected, despite the fact that the only available treatments are highly toxic drugs. In this work, we have isolated, through bioguided assays, the major antileishmanial compounds of the toad
Rhinella jimi parotoid macrogland secretion. Mass spectrometry and
1H and
13C NMR spectroscopic analyses were able to demonstrate that the active molecules are
telocinobufagin and
hellebrigenin. Both steroids demonstrated activity against
Leishmania (L.)
chagasi promastigotes, but only hellebrigenin was active against
Trypanosoma cruzi trypomastigotes. These steroids were active against the intracellular amastigotes of
Leishmania, with no activation of nitric oxide production by macrophages. Neither cytotoxicity against mouse macrophages nor hemolytic activities were observed. The ultrastructural studies with promastigotes revealed the induction of mitochondrial damage and plasma membrane disturbances by telocinobufagin, resulting in cellular death. This novel biological effect of
R. jimi steroids could be used as a template for the design of new therapeutics against Leishmaniasis and American Trypanosomiasis.
Tropical diseases caused by parasitic infections continue to cause socioeconomic distress worldwide. Among these, Chagas disease has become a great concern because of globalization. Caused by ...Trypanosoma cruzi, there is an increasing need to discover new, more effective methods to manage infections that minimize disease onset. Antimicrobial peptides represent a possible solution to this challenge. As effector molecules of the innate immune response against pathogens, they are the first line of defense found in all multi-cellular organisms. In amphibians, temporins are a large family of antimicrobial peptides found in skin secretions. Their functional roles and modes of action present unique properties that indicate possible candidates for therapeutic applications. Here, we investigated the trypanocide activity of temporizin and temporizin-1. Temporizin is an artificial, hybrid peptide containing the N-terminal region of temporin A, the pore-forming region of gramicidin and a C-terminus consisting of alternating leucine and lysine. Temporizin-1 is a modification of temporizin with a reduction in the region responsible for insertion into membranes. Their activities were evaluated in a cell permeabilization assay by flow cytometry, an LDH release assay, electron microscopy, an MTT assay and patch clamp experiments. Both temporizin and temporizin-1 demonstrated toxicity against T. cruzi with temporizin displaying slightly more potency. At concentrations up to 100 μg/ ml, both peptides exhibited low toxicity in J774 cells, a macrophage lineage cell line, and no toxicity was observed in mouse primary peritoneal macrophages. In contrast, the peptides showed some toxicity in rat adenoma GH3 cells and Jurkat human lymphoma cells with temporizin-1 displaying lower toxicity. In summary, a shortened form of the hybrid temporizin peptide, temporizin-1, was efficient at killing T. cruzi and it has low toxicity in wild-type mammalian cells. These data suggest that temporizin-1 might be a candidate for Chagas disease therapy.
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