Number Of Viable Parasites Research Articles

Trypanocidal potential of synthetic p-aminochalcones: In silico and in vitro evaluation

Chagas disease (CD) is a neglected tropical disease of worldwide health concern, caused by the flagellate protozoan Trypanosoma cruzi (T. cruzi), endemic in Latin America and present in North America and Europe. The WHO recommended drug for CD, benznidazole has low safety profile and several limitations. Therefore, an entity with better therapeutic potential to treat CD is required. Chalcones are an important class of compounds, which have shown antichagasic potential. Thus, the objective of this study was to evaluate the activity of synthetic p-aminochalcones against T. cruzi. Chalcones 1 and 2 were synthesized by Claisen-Schmidt condensation and characterized by both spectroscopic and theoretical methods. Initially, they were submitted to molecular docking simulations using cruzain and trypanothione reductase (TR) enzymes. It was expected to observe the possible interactions of chalcones with the catalytic site and other important regions of these main pharmacological targets of T. cruzi. Their cytotoxicity within host cells were assessed by MTT reduction assay using LLC-MK2 cells, with CC50 = 85.6 ± 9.2 µM and 1115 ± 381.7 µM for chalcones 1 and 2, respectively. These molecules were also tested against epimastigote and trypomastigote life forms of T. cruzi, causing reduction in the number of viable parasites. For the evaluation of the effect on intracellular amastigotes, infected LLC-MK2 cells were incubated with the chalcones for 24 h, causing reduction in the percentage of infected cells and the number of amastigotes/100 cells. Finally, flow cytometry assays were performed for analyzing cell death mechanisms (7-AAD/AxPE labelling), cytoplasmic ROS accumulation (DCFH-DA assay) and mitochondrial transmembrane potential disruption (Rho123 assay). Both chalcones (1 and 2) caused membrane damage, ROS accumulation and mitochondrial depolarization. In conclusion, the synthetic p-aminochalcones presented trypanocidal effect, causing membrane damage and oxidative stress. Their mechanism of action may be related to cruzain and TR inhibition.

Overexpression of Leishmania major protein arginine methyltransferase 6 reduces parasite infectivity in vivo

Arginine methylation is catalysed by Protein Arginine Methyltransferases (PRMTs) and can affect how a target protein functions and how it interacts with other macromolecules, which in turn impacts on cell metabolism and gene expression control. Leishmania parasites express five different PRMTs, and although the presence of each individual PRMT is not essential per se, the imbalanced activity of these PRMTs can impact the virulence of Leishmania parasites in vitro and in vivo. Here we created a Leishmania major cell line overexpressing PRMT6 and show that similar to what was observed for the T. brucei homologous enzyme, L. major PRMT6 probably has a narrow substrate range. However, its overexpression notably impairs the infection in mice, with a mild reduction in the number of viable parasites in the lymph nodes. Our results indicate that arginine methylation by LmjPRMT6 plays a significant role in the adaptation of the parasite to the environment found in the mammalian host.

Predicting Optimal Antimalarial Drug Combinations from a Standardized Plasmodium falciparum Humanized Mouse Model.

The development of new combinations of antimalarial drugs is urgently needed to prevent the spread of parasites resistant to drugs in clinical use and contribute to the control and eradication of malaria. In this work, we evaluated a standardized humanized mouse model of erythrocyte asexual stages of Plasmodium falciparum (PfalcHuMouse) for the selection of optimal drug combinations. First, we showed that the replication of P. falciparum was robust and highly reproducible in the PfalcHuMouse model by retrospective analysis of historical data. Second, we compared the relative value of parasite clearance from blood, parasite regrowth after suboptimal treatment (recrudescence), and cure as variables of therapeutic response to measure the contributions of partner drugs to combinations in vivo. To address the comparison, we first formalized and validated the day of recrudescence (DoR) as a new variable and found that there was a log-linear relationship with the number of viable parasites per mouse. Then, using historical data on monotherapy and two small cohorts of PfalcHuMice evaluated with ferroquine plus artefenomel or piperaquine plus artefenomel, we found that only measurements of parasite killing (i.e., cure of mice) as a function of drug exposure in blood allowed direct estimation of the individual drug contribution to efficacy by using multivariate statistical modeling and intuitive graphic displays. Overall, the analysis of parasite killing in the PfalcHuMouse model is a unique and robust experimental in vivo tool to inform the selection of optimal combinations by pharmacometric pharmacokinetic and pharmacodynamic (PK/PD) modeling.

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195

BACKGROUNDLeishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent.OBJECTIVETo evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro.METHODSThe viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ.FINDINGSMedian IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195.MAIN CONCLUSIONSPK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.

Miltefosine Increases Lipid and Protein Dynamics in Leishmania amazonensis Membranes at Concentrations Similar to Those Needed for Cytotoxicity Activity

Miltefosine (MT) is a membrane-active alkylphospholipid licensed for the topical treatment of breast cancer skin metastases and the oral treatment of leishmaniasis, although its mechanism of action remains unclear. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled lipid and a thiol-specific spin label in the plasma membrane of Leishmania promastigotes showed that MT causes dramatic increases in membrane dynamics. Although these alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture.

Photodynamic antimicrobial chemotherapy (PACT) using phenothiazine derivatives as photosensitizers against Leishmania braziliensis

Cutaneous and mucocutaneous leishmaniasis are diseases characterized by skin or mucosal manifestations. In the new world, Leishmania braziliensis is the main etiological agent of cutaneous leishmaniasis, condition that may evolve to the mucocutaneous form. The therapeutic arsenal routinely employed to treat infected patients is unsatisfactory, especially for pentavalent antimonials, treatment recommended by the WHO, as they are often highly toxic, poorly tolerated and of variable effectiveness. This work aimed to evaluate in vitro the effectiveness of photodynamic antimicrobial chemotherapy as a new approach for the treatment of leishmaniasis. A laser (λ = 660 nm, 40 mW, 4.2 J/cm2 , and 8.4 J/cm2 , CW) associated to phenothiazine's derivatives (5 and 10 µg/ml, toluidine blue O, methylene blue, or phenothiazine) on the promastigote forms of L. braziliensis in a single session. Samples were removed and analyzed in a hemocytometer 72 hours after PACT and viability of the parasites was assessed in quadruplicates. An important decrease in the number of viable parasites on all treated groups in comparison to their controls was observed as all tested compounds lead to significant parasite lethality being the highest lethality achieved with 10 µg/ml of TBO. No lethality was observed on groups treated with laser or with any of the compounds separately. TBO presented higher parasite lethality in comparison to MB with impressive reduction from 1 hour to 5 minutes of pre-incubation time.

Exacerbation of Leishmania (Viannia) shawi infection in BALB/c mice after immunization with soluble antigen from amastigote forms

The present study aimed to evaluate the effects of immunization with soluble amastigote (AmaAg) and promastigote (ProAg) antigens from Leishmania (Viannia) shawi on the course of infection in BALB/c mice. After immunization with AmaAg, the challenged group showed greater lesion size and parasite load in the skin and lymph nodes, associated with diminished interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-γ and nitrate levels in the supernatant of lymph node cell cultures, together with increases in transforming growth factor (TGF)-β concentrations and humoral immune response. In contrast, immunization with ProAg led to smaller lesion size with reduced numbers of viable parasites in the skin. Protection was associated with increases in IL-12, IFN-γ, TGF-β and nitrates and decreases in IL-4 and IL-10 levels. Concerning humoral immune response, a significant reduction in anti-leishmania immunoglobulin G was verified in the ProAg-challenged group. Analysis of these results suggests that AmaAg induced a suppressive cellular immune response in mice, favouring the spread of infection, whereas ProAg induced partial protection associated with increased cellular immune response.

Effect of the antimicrobial peptide, D-hecate, on trichomonads.

Tritrichomonas foetus and Trichomonas vaginalis are protozoan parasites that cause sexually transmitted diseases in cattle and humans, respectively. There is a need for new antimicrobial agents to treat or prevent trichomoniasis because there are currently no approved chemotherapeutic agents against T. foetus and resistance of T. vaginalis to metronidazole does occur. Therefore, we evaluated the effect of a novel antimicrobial peptide, D-hecate, on the viability of 6 isolates of T. foetus and T. vaginalis in vitro. Tritrichomonas foetus and T. vaginalis were grown to mid log phase (24 hr) or late log/stationary phase (48 hr). Parasites at 10(6)/ml were mixed with equal volumes of D-hecate to final concentrations of 10 microM, 20 microM. and 40 microM of D-hecate. Controls had minimal essential medium (MEM) alone. The numbers of viable parasites were determined microscopically after 10, 20, and 30 min of incubation at 37 C with D-hecate or MEM. Our results show that D-hecate killed all 6 isolates of T. foetus and T. vaginalis evaluated. The killing effect was dependent on the concentration of the peptide, incubation time, and phase of growth of the parasites. Ultrastructural studies of parasites treated with 10 microM of D-hecate revealed extensive damage to the plasma membrane of most T. foetus and T. vaginalis cells, while a few cells were distorted but remained intact. D-Hecate may be a useful chemotherapeutic agent for the treatment of trichomoniasis.

Therapeutic effect of oral Kalanchoe pinnata leaf extract in murine leishmaniasis

The effect of a leaf extract from Kalanchoe pinnata (Kp) was investigated in BALB/c mice infected with Leishmania amazonensis. Oral treatment with Kp significantly delayed onset of disease as compared to untreated mice or mice receiving Kp by the intravenous or topical routes. When initiated at early stages of infection, daily oral doses of 8 mg prevented lesion growth and the effect was long-lasting, comparable to the reference antileishmanial drug Glucantime. The decreased lesion growth using the oral route was accompanied by a significant decrease in the number of viable parasites. Protection was accompanied by a diminished capacity of animals to develop delayed-type hypersensitivity and to produce specific antibodies.