Drug screening and development cascade for Chagas disease: an update of in vitro and in vivo experimental models.

Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years.

Bone marrow-derived mesenchymal stem/stromal cells reverse the sensorial diabetic neuropathy via modulation of spinal neuroinflammatory cascades.

BACKGROUND: Diabetic neuropathy (DN) is a frequent and debilitating manifestation of diabetes mellitus, to which there are no effective therapeutic approaches. Mesenchymal stem/stromal cells (MSC) have a great potential for the treatment of this syndrome, possibly through regenerative actions on peripheral nerves. Here, we evaluated the therapeutic effects of MSC on spinal neuroinflammation, as well as on ultrastructural aspects of the peripheral nerve in DN-associated sensorial dysfunction. METHODS: C57Bl/6 mice were treated with bone marrow-derived MSC (1 × 106), conditioned medium from MSC cultures (CM-MSC) or vehicle by endovenous route following the onset of streptozotocin (STZ)-induced diabetes. Paw mechanical and thermal nociceptive thresholds were evaluated by using von Frey filaments and Hargreaves test, respectively. Morphological and morphometric analysis of the sciatic nerve was performed by light microscopy and transmission electron microscopy. Mediators and markers of neuroinflammation in the spinal cord were measured by radioimmunoassay, real-time PCR, and immunofluorescence analyses. RESULTS: Diabetic mice presented behavioral signs of sensory neuropathy, mechanical allodynia, and heat hypoalgesia, which were completely reversed by a single administration of MSC or CM-MSC. The ultrastructural analysis of the sciatic nerve showed that diabetic mice exhibited morphological and morphometric alterations, considered hallmarks of DN, such as degenerative changes in axons and myelin sheath, and reduced area and density of unmyelinated fibers. In MSC-treated mice, these structural alterations were markedly less commonly observed and/or less pronounced. Moreover, MSC transplantation inhibited multiple parameters of spinal neuroinflammation found in diabetic mice, causing the reduction of activated astrocytes and microglia, oxidative stress signals, galectin-3, IL-1ß, and TNF-α production. Conversely, MSC increased the levels of anti-inflammatory cytokines, IL-10, and TGF-ß. CONCLUSIONS: The present study described the modulatory effects of MSC on spinal cord neuroinflammation in diabetic mice, suggesting new mechanisms by which MSC can improve DN.

The biochemical characterization, stabilization studies and the antiproliferative effect of bromelain against B16F10 murine melanoma cells.

The current study aims to extract bromelain from different parts (stem, crown, peels, pulp and leaves) of Ananas comosus var. comosus AGB 772; to determine of optimum pH and temperature; to test bromelain stability in disodium EDTA and sodium benzoate, and to investigate its pharmacological activity on B16F10 murine melanoma cells in vitro. The highest enzymatic activity was found in bromelain extracted from the pulp and peel. The optimum bromelain pH among all studied pineapple parts was 6.0. The optimum temperature was above 50 °C in all bromelain extracts. The fluorescence analysis confirmed the stability of bromelain in the presence of EDTA and sodium benzoate. Bromelain was pharmacologically active against B16F10 melanoma cells and it was possible verifying approximately 100% inhibition of tumor cell proliferation in vitro. Since bromelain activity was found in different parts of pineapple plants, pineapple residues from the food industry may be used for bromelain extraction.

Cell Death of P. vivax Blood Stages Occurs in Absence of Classical Apoptotic Events and Induces Eryptosis of Parasitized Host Cells.

Elucidation of pathways regulating parasite cell death is believed to contribute to identification of novel therapeutic targets for protozoan diseases, and in this context, apoptosis-like cell death has been reported in different groups of protozoa, in which metacaspases seem to play a role. In the genus Plasmodium, apoptotic markers have been detected in P. falciparum and P. berghei, and no study focusing on P. vivax cell death has been reported so far. In the present study, we investigated the susceptibility of P. vivax to undergo apoptotic cell death after incubating mature trophozoites with the classical apoptosis inducer staurosporine. As assessed by flow cytometry assays, staurosporine inhibited parasite intraerythrocytic development, which was accompanied by a decrease in cell viability, evidenced by reduced plasmodial mitochondrial activity. However, typical signs of apoptosis, such as DNA fragmentation, chromatin condensation, and nuclear segregation, were not detected in the parasites induced to cell death, and no significant alteration in metacaspase gene expression (PvMCA1) was observed under cell death stimulus. Interestingly, dying parasites positively modulated cell death (eryptosis) of host erythrocytes, which was marked by externalization of phosphatidylserine and cell shrinkage. Our study shows for the time that P. vivax blood stages may not be susceptible to apoptosis-like processes, while they could trigger eryptosis of parasitized cells by undergoing cell death. Further studies are required to elucidate the cellular machinery involved in cell death of P. vivax parasites as well as in the modulation of host cell death.

The repositioned drugs disulfiram/diethyldithiocarbamate combined to benznidazole: Searching for Chagas disease selective therapy, preventing toxicity and drug resistance.

Chagas disease (CD) affects at least 6 million people in 21 South American countries besides several thousand in other nations all over the world. It is estimated that at least 14,000 people die every year of CD. Since vaccines are not available, chemotherapy remains of pivotal relevance. About 30% of the treated patients cannot complete the therapy because of severe adverse reactions. Thus, the search for novel drugs is required. Here we tested the benznidazole (BZ) combination with the repositioned drug disulfiram (DSF) and its derivative diethyldithiocarbamate (DETC) upon Trypanosoma cruzi in vitro and in vivo. DETC-BZ combination was synergistic diminishing epimastigote proliferation and enhancing selective indexes up to over 10-fold. DETC was effective upon amastigotes of the BZ- partially resistant Y and the BZ-resistant Colombiana strains. The combination reduced proliferation even using low concentrations (e.g., 2.5 µM). Scanning electron microscopy revealed membrane discontinuities and cell body volume reduction. Transmission electron microscopy revealed remarkable enlargement of endoplasmic reticulum cisternae besides, dilated mitochondria with decreased electron density and disorganized kinetoplast DNA. At advanced stages, the cytoplasm vacuolation apparently impaired compartmentation. The fluorescent probe H2-DCFDA indicates the increased production of reactive oxygen species associated with enhanced lipid peroxidation in parasites incubated with DETC. The biochemical measurement indicates the downmodulation of thiol expression. DETC inhibited superoxide dismutase activity on parasites was more pronounced than in infected mice. In order to approach the DETC effects on intracellular infection, peritoneal macrophages were infected with Colombiana trypomastigotes. DETC addition diminished parasite numbers and the DETC-BZ combination was effective, despite the low concentrations used. In the murine infection, the combination significantly enhanced animal survival, decreasing parasitemia over BZ. Histopathology revealed that low doses of BZ-treated animals presented myocardial amastigote, not observed in combination-treated animals. The picrosirius collagen staining showed reduced myocardial fibrosis. Aminotransferase de aspartate, Aminotransferase de alanine, Creatine kinase, and urea plasma levels demonstrated that the combination was non-toxic. As DSF and DETC can reduce the toxicity of other drugs and resistance phenotypes, such a combination may be safe and effective.

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes.

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC50 = 28.5 ± 2.0 µM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.

Trichoderma stromaticum spores induce autophagy and downregulate inflammatory mediators in human peripheral blood-derived macrophages.

Trichoderma spp. are usually considered safe and normally used as biocontrol and biofertilization. Safety for human health is evaluated by several tests that detect various effects such as allergenicity, toxicity, infectivity, and pathogenicity. However, they do not evaluate the effects of the agent upon the immune system. The aim of this study was to investigate the interaction between T. stromaticum spores and mammalian cells to assess the immunomodulatory potential of the spores of this fungus. First, mouse macrophage cell line J774 and human macrophages were exposed to fungal spores and analyzed for structural features, through scanning and transmission electron microscopy. Then, various analysis were performed in human macrophages as to their effect in some functional and molecular aspects of the immune system through immunocytochemistry, flow cytometry and gene expression assays. We demonstrated that T. stromaticum spores induces autophagy and autophagy-related genes (ATGs) and downmodulate inflammatory mediators, including ROS, NLRP3, the cytokines IL-1ß, IL-18, IL-12 and IL-10, as well as TLR2, TLR4, miR-146b and miR-155, which may lead to an augmented susceptibility to pathogens. Our study shows the extension of damages the biofungicide Tricovab® can cause in the innate immune response. Further studies are necessary to elucidate other innate and adaptive immune responses and, consequently, the safety of this fungus when in contact with humans.

Phagocytosis is inhibited by autophagic induction in murine macrophages.

Recent studies have demonstrated that communication takes place between the autophagic and phagocytic pathways, indicating that the convergence of these two pathways plays an important role in the innate immune response against intracellular microbes. The present study investigated the effect of autophagic induction on the phagocytic capacity of murine macrophages. Autophagy induced by physiological and pharmacological means was shown to reduce the phagocytic capacity of murine macrophages, regardless of cell origin or the nature of the phagocytosed particles themselves. This autophagic inhibitory effect on phagocytosis was shown to be an early and reversible event that results in no loss of cell viability. Furthermore, the data presented herein demonstrate that the induction of autophagy does not affect a macrophage's capacity to recognize and bind to particles, indicating that autophagy does not inhibit the particle recognition process, even though particle internalization is suppressed. The findings herein support the notion that phagocytosis and autophagy may be interdependent and complementary processes.

Leishmania amazonensis: characterization of an ecto-3'-nucleotidase activity and its possible role in virulence.

Ecto-3'-nucleotidase/nuclease (3'NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3'mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3'-nucleotidase activity (La3'-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3'-nucleotidase inhibitor and approach the possible involvement of ecto-3'-nucleotidase in cellular adhesion. La3'-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3'-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu(2+) ions. Interestingly, ecto-3'-nucleotidase activity is 60-fold higher than that of ecto-5'-nucleotidase in L. amazonensis. Additionally, ecto-3'-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage-parasite attachment/invasion was increased by 400% in the presence of adenosine 3'-monophosphate (3'AMP); however, this effect was reverted by TTM treatment. We believe that La3'-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.

Ethanolic Extract of the Fungus Trichoderma asperelloides Induces Ultrastructural Effects and Death on Leishmania amazonensis.

The Trichoderma genus comprises several species of fungi whose diversity of secondary metabolites represents a source of potential molecules with medical application. Because of increased pathogen resistance and demand for lower production costs, the search for new pharmacologically active molecules effective against pathogens has become more intense. This is particularly evident in the case of American cutaneous leishmaniasis due to the high toxicity of current treatments, parenteral administration, and increasing rate of refractory cases. We have previously shown that a fungus from genus Trichoderma can be used for treating cerebral malaria in mouse models and inhibit biofilm formation. Here, we evaluated the effect of the ethanolic extract of Trichoderma asperelloides (Ext-Ta) and its fractions on promastigotes and amastigotes of Leishmania amazonensis, a major causative agent of cutaneous leishmaniasis in the New World. Ext-Ta displayed leishmanicidal action on L. amazonensis parasites, and its pharmacological activity was associated with the low-molecular-weight fraction (LMWF) of Ext-Ta. Ultrastructural analysis demonstrated morphological alterations in the mitochondria and the flagellar pocket of promastigotes, with increased lipid body and acidocalcisome formation, microtubule disorganization of the cytoplasm, and intense vacuolization of the cytoplasm when amastigotes were present. We suggest the antiparasitic activity of Trichoderma fungi as a promising tool for developing chemotherapeutic leishmanicidal agents.

Histone deacetylases inhibitors as new potential drugs against Leishmania braziliensis, the main causative agent of new world tegumentary leishmaniasis.

The protozoan parasite Leishmania braziliensis is a major causative agent of the neglected tropical diseases Cutaneous and Mucocutaneous Leishmaniases in the New World. There are no vaccines to prevent the infection and the treatment relies on few drugs that often display high toxicity and costs. Thus, chemotherapeutic alternatives are required. Histone Deacetylases (HDACs) are epigenetic enzymes involved in the control of chromatin structure. In this work, we tested an in-house library of 78 hydroxamic acid derivatives as putative inhibitors of L. braziliensis HDACs (HDACi). The compounds were evaluated in relation to the toxicity to the host cell macrophage and to the leishmanicidal effect against L. braziliensis during in vitro infection. Eight HDACi showed significant leishmanicidal effects and the top 5 compounds showed effective concentrations (EC50) in the range of 4.38 to 10.21 µM and selectivity indexes (SI) from of 6 to 21.7. Analyses by Transmission Electron Microscopy (TEM) indicated induction of apoptotic cell death of L. braziliensis amastigotes with a necrotic phenotype. An altered chromatin condensation pattern and cellular disorganization of intracellular amastigotes was also observed. A tight connection between the mitochondrion and nuclear protrusions, presumably of endoplasmic reticulum origin, was found in parasites but not in the host cell. In flow cytometry (FC) analyses, HDACi promoted parasite cell cycle arrest in the G2-M phase and no changes were found in macrophages. In addition, the direct effect of HDACi against the promastigotes showed apoptosis as the main mechanism of cell death. The FC results corroborate the TEM analyses indicating that the HDACi lead to changes in the cell cycle and induction of apoptosis of L. braziliensis. The production of nitric oxide by the infected macrophages was not altered after treatment with the top 5 compounds. Taken together, our results evidenced new HDACi as promising agents for the development of new treatments for American Tegumentary Leishmaniasis caused by L. braziliensis.

PEMF fails to enhance nerve regeneration after sciatic nerve crush lesion.

The use of electromagnetic fields has been reported to enhance peripheral nerve regeneration. This study aimed to identify the effects of a prolonged protocol of low-frequency pulsed electromagnetic field (PEMF) on peripheral nerve regeneration. Thirty-four male Swiss mice (Mus musculus) were divided into PEMF (n = 17) and control (n = 17) groups. All animals underwent a unilateral sciatic-crush lesion, and the PEMF group was exposed to a 72-Hz, 2-G electromagnetic field for 30 min, five days a week, for three weeks. Functional analysis was carried out weekly. After three weeks, the animals were euthanized, and histological, morphometric, oxidative stress, and TGF-beta1 analyses were performed. Functional analysis showed no differences between the groups. Histological appearance was similar between PEMF and control nerves. Morphometric assessment showed that the PEMF nerves trended toward decreased regeneration. The levels of free radicals were more pronounced in PEMF nerves, but were not associated with an increase in the content of the TGF-beta1/Smad signaling pathway. Prolonged PEMF regimen leads to delayed histological peripheral nerve regeneration and increased oxidative stress but no loss of function recovery.

Synthesis of cinnamic acid derivatives and leishmanicidal activity against Leishmania braziliensis.

Leishmania braziliensis is one of the pathogenic agents of cutaneous and mucocutanoeous leishmaniasis. There are no validated vaccines to prevent the infection and the treatment relies on drugs that often present severe side effects, which justify the efforts to find new potential antileishmanial drugs. An alternative to promote the discovery of new drugs would be the association of different chemical groups of bioactive compounds. Here we describe the synthesis and bioactivity evaluation against L. braziliensis of cinnamic acid derivatives possessing isobenzofuranone and 1,2,3-triazole functionalities. We tested 25 compounds at 10 µM concentration against extracellular promastigotes and intracellular amastigotes during macrophage infection. Most compounds were more active against amastigotes than to promastigotes. The derivatives (E)-3-oxo-1,3-dihydroisobenzofuran-5-yl-(3,4,5-trimethoxy) cinnamate (5c), (1-(3,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9g), and (1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl cinnamate (9l) were the most effective presenting over 80% toxicity on L. braziliensis amastigotes. While compound 5c is a cinnamate with an isobenzofuranone portion, 9g and 9l are triazolic cinnamic acid derivatives. The action of these compounds was comparable to amphotericin B used as positive control. Ultrastructural analysis revealed that 5c-treated parasites showed impaired cytokinesis and apoptosis triggering. Taken together, these results highlight the potential of cinnamic acid derivatives in development of novel anti-leishmanial drugs.

Drug screening and development cascade for Chagas disease: an update of in vitro and in vivo experimental models

Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years.

A new approach to assess function after sciatic nerve lesion in the mouse - adaptation of the sciatic static index.

Among the numerous ways of assessing regeneration after peripheral nerve lesions, the analysis of gait is one of the most important, because it shows the recovery of function, which is the ultimate goal of the repair machinery. The sciatic function index was introduced as a method to assess reinnervation after an experimental sciatic nerve lesion, and was adapted to the mouse model. The sciatic static index (SSI), is more simple and practical to perform, and is not so influenced by gait's velocity, but this method has not yet been adapted to the mouse model of sciatic lesion. We used 63 male Swiss mice (Mus musculus) to develop a formula to the sciatic static index in mice (SSIm). The animals were divided on three groups (control, transection and crush). They were evaluated at the preoperative and 7th, 14th, 21st, 28th, 35th and 42nd days postoperative by the ink track method (SFI), and by the acquisition of photographs of the plantar aspects of the injured and uninjured hind paws. The parameters evaluated were the 1-5 toe spread (TS), the 2-4 toe spread (ITS) and the distance between the tip of the third toe and the most posterior aspect of the paw (PL), on both methods. After verifying the temporal pattern of function, correlation and reproducibility of the measurements, we performed a multiple regression analysis using SFI values as dependent variable, and the TS, ITS and PL measured with the photo method as independent variables, and found the formula of the SSI for mice (SSIm). The three groups (control, transection and crush) had a characteristic pattern of dysfunction. The parameters measured in the ink and photo method had variable but significant correlations between them (P<0.000), but photo method of measurement showed a better reproducibility. The correlation between SFI and SSIm showed a high correlation coefficient (r=0.892, P<0.000), and demonstrates that SSIm can be used as an alternative method to assess the functional status relative of sciatic nerve activity in mice.

Plants and intestinal parasitosis: a review on ethnopharmacological use by the Kantaruré-Batida indigenous community of Brazil / Plantas y parasitosis intestinal: una revisión sobre el uso etnofarmacológico de la comunidad indígena Kantaruré-Batida de Brasil

Indigenous knowledge is one of the most notable traditional sources about plants used to treat diseases. Thus, the aim of this study was to describe the botanical features and to investigate the pharmacological properties of plants used by the Kantaruré-Batida community to treat intestinal parasitosis. Botanical characterization was performed based on integrative review andon access to 'Flora do Brasil' platform, whereas plants' pharmacological properties were investigated through systematic review. Among the 21 ethnospecies used, 48% were described as having activity against intestinal parasites. Although 52% had no such activity described, other properties can account for their use, that indicates that further studies are required. Therefore, this review reinforces the importance of valuing indigenous knowledge as tool to guide antiparasitic agent trials.

El conocimiento indígena es una de las fuentes tradicionales más notables sobre las plantas utilizadas para tratar enfermedades. Por lo tanto, el objetivo de este estudio fue describir las características botánicas e investigar las propiedades farmacológicas de las plantas utilizadas por la comunidad Kantaruré-Batida para tratar la parasitosis intestinal. La caracterización botánica se realizó con base en una revisión integradora y en el acceso a la plataforma "Flora do Brasil", mientras que las propiedades farmacológicas de las plantas se investigaron mediante una revisión sistemática. Entre las 21 etnoespecies utilizadas, se describió que el 48% tenía actividad contra los parásitos intestinales. Aunque el 52% no tenía tal actividad descrita, otras propiedades pueden explicar su uso, lo que indica que se requieren más estudios. Por lo tanto, esta revisión refuerza la importancia de valorar el conocimiento indígena como herramienta para guiar los ensayos de agentes antiparasitarios.

Effects of a novel ß-lapachone derivative on Trypanosoma cruzi: Parasite death involving apoptosis, autophagy and necrosis.

Natural products comprise valuable sources for new antiparasitic drugs. Here we tested the effects of a novel ß-lapachone derivative on Trypanosoma cruzi parasite survival and proliferation and used microscopy and cytometry techniques to approach the mechanism(s) underlying parasite death. The selectivity index determination indicate that the compound trypanocidal activity was over ten-fold more cytotoxic to epimastigotes than to macrophages or splenocytes. Scanning electron microscopy analysis revealed that the R72 ß-lapachone derivative affected the T. cruzi morphology and surface topography. General plasma membrane waving and blebbing particularly on the cytostome region were observed in the R72-treated parasites. Transmission electron microscopy observations confirmed the surface damage at the cytostome opening vicinity. We also observed ultrastructural evidence of the autophagic mechanism termed macroautophagy. Some of the autophagosomes involved large portions of the parasite cytoplasm and their fusion/confluence may lead to necrotic parasite death. The remarkably enhanced frequency of autophagy triggering was confirmed by quantitating monodansylcadaverine labeling. Some cells displayed evidence of chromatin pycnosis and nuclear fragmentation were detected. This latter phenomenon was also indicated by DAPI staining of R72-treated cells. The apoptotis induction was suggested to take place in circa one-third of the parasites assessed by annexin V labeling measured by flow cytometry. TUNEL staining corroborated the apoptosis induction. Propidium iodide labeling indicate that at least 10% of the R72-treated parasites suffered necrosis within 24 h. The present data indicate that the ß-lapachone derivative R72 selectively triggers T. cruzi cell death, involving both apoptosis and autophagy-induced necrosis.

The Effectiveness of Natural Diarylheptanoids against Trypanosoma cruzi: Cytotoxicity, Ultrastructural Alterations and Molecular Modeling Studies.

Curcumin (CUR) is the major constituent of the rhizomes of Curcuma longa and has been widely investigated for its chemotherapeutic properties. The well-known activity of CUR against Leishmania sp., Trypanosoma brucei and Plasmodium falciparum led us to investigate its activity against Trypanosoma cruzi. In this work, we tested the cytotoxic effects of CUR and other natural curcuminoids on different forms of T. cruzi, as well as the ultrastructural changes induced in epimastigote form of the parasite. CUR was verified as the curcuminoid with more significant trypanocidal properties (IC50 10.13 µM on epimastigotes). Demethoxycurcumin (DMC) was equipotent to CUR (IC50 11.07 µM), but bisdemethoxycurcumin (BDMC) was less active (IC50 45.33 µM) and cyclocurcumin (CC) was inactive. In the experiment with infected murine peritoneal macrophages all diarylheptanoids were more active than the control in the inhibition of the trypomastigotes release. The electron microscopy images showed ultrastructural changes associated with the cytoskeleton of the parasite, indicating tubulin as possible target of CUR in T. cruzi. The results obtained by flow cytometry analysis of DNA content of the parasites treated with natural curcuminoids suggested a mechanism of action on microtubules related to the paclitaxel`s mode of action. To better understand the mechanism of action highlighted by electron microscopy and flow cytometry experiments we performed the molecular docking of natural curcuminoids on tubulin of T. cruzi in a homology model and the results obtained showed that the observed interactions are in accordance with the IC50 values found, since there CUR and DMC perform similar interactions at the binding site on tubulin while BDMC do not realize a hydrogen bond with Lys163 residue due to the absence of methoxyl groups. These results indicate that trypanocidal properties of CUR may be related to the cytoskeletal alterations.

Structural and morphological characterization of hemozoin produced by Schistosoma mansoni and Rhodnius prolixus.

Hemozoin (Hz) is a heme crystal produced upon the digestion of hemoglobin (Hb) by blood-feeding organisms as a main mechanism of heme disposal. The structure of Hz consists of heme dimers bound by reciprocal iron-carboxylate interactions and stabilized by hydrogen bonds. We have recently described heme crystals in the blood fluke, Schistosoma mansoni, and in the kissing bug, Rhodnius prolixus. Here, we characterized the structures and morphologies of the heme crystals from those two organisms and compared them to synthetic beta-hematin (betaH). Synchrotron radiation X-ray powder diffraction showed that all heme crystals share the same unit cell and structure. The heme crystals isolated from S. mansoni and R. prolixus consisted of very regular units assembled in multicrystalline spherical structures exhibiting remarkably distinct surface morphologies compared to betaH. In both organisms, Hz formation occurs inside lipid droplet-like particles or in close association to phospholipid membranes. These results show, for the first time, the structural and morphological characterization of natural Hz samples obtained from these two blood-feeding organisms. Moreover, Hz formation occurring in close association to a hydrophobic environment seems to be a common trend for these organisms and may be crucial to produce very regular shaped phases, allowing the formation of multicrystalline assemblies in the guts of S. mansoni and R. prolixus.

The parameters of transcutaneous electrical nerve stimulation are critical to its regenerative effects when applied just after a sciatic crush lesion in mice.

We investigated the effect of two frequencies of transcutaneous electrical nerve stimulation (TENS) applied immediately after lesion on peripheral nerve regeneration after a mouse sciatic crush injury. The animals were anesthetized and subjected to crushing of the right sciatic nerve and then separated into three groups: nontreated, Low-TENS (4 Hz), and High-TENS (100 Hz). The animals of Low- and High-TENS groups were stimulated for 2 h immediately after the surgical procedure, while the nontreated group was only positioned for the same period. After five weeks the animals were euthanized, and the nerves dissected bilaterally for histological and histomorphometric analysis. Histological assessment by light and electron microscopy showed that High-TENS and nontreated nerves had a similar profile, with extensive signs of degeneration. Conversely, Low-TENS led to increased regeneration, displaying histological aspects similar to control nerves. High-TENS also led to decreased density of fibers in the range of 6-12 µm diameter and decreased fiber diameter and myelin area in the range of 0-2 µm diameter. These findings suggest that High-TENS applied just after a peripheral nerve crush may be deleterious for regeneration, whereas Low-TENS may increase nerve regeneration capacity.