Effect of DODAB Nano-Sized Cationic Bilayer Fragments against Leishmania amazonensis.

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 Leishmania amazonensis, 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 (EC50) 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.

Preclinical Investigation of Methylene Blue-mediated Antimicrobial Photodynamic Therapy on Leishmania Parasites Using Real-Time Bioluminescence.

Cutaneous leishmaniasis (CL) is a neglected disease that promotes destructive lesions. Difficulties in treatment are related to accessibility of drugs, resistance and toxicity. Antimicrobial photodynamic therapy (APDT) has been emerging as a promising treatment for CL. In this work, we evaluated methylene blue (MB)-mediated APDT (MB-APDT) on Leishmania amazonensis in vitro and in vivo by bioluminescence technique. In vitro, MB-APDT was performed using a red LED (λ = 660 ± 11 nm, 100 mW cm-2 ) and MB (100 µm) at different light doses. In vivo, mice were infected and 4 weeks later, randomly divided into three groups: control, APDT 1 (single session) and APDT 2 (two sessions of MB-APDT). MB was used at 100 µm and energy dose was established at 150 J cm-2 . Parasite burden, lesion size and pain were evaluated weekly for 4 weeks. In vitro, lethal dose for 90% parasite inactivation was achieved at 48.8 J cm-2 . In vivo, although APDT 1 and APDT 2 groups have showed similar parasite burden after 4 weeks, two sessions were clinically better, especially considering the inflammatory process associated to CL. Our findings reinforce MB-APDT as a cost-effective treatment to combat CL.

Nitric oxide-loaded chitosan nanoparticles as an innovative antileishmanial platform.

Leishmaniasis is a neglected tropical disease that demands for new therapeutic strategies due to adverse side effects and resistance development promoted by current drugs. Nitric oxide (NO)-donors show potential to kill Leishmania spp. but their use is limited because of their instability. In this work, we synthesize, characterize, and encapsulate S-nitroso-mercaptosuccinic acid into chitosan nanoparticles (NONPs) and investigate their activity on promastigotes and intracellular amastigotes of Leishmania (Leishmania) amazonensis. Cytotoxicity on macrophages was also evaluated. We verified that NONPs reduced both forms of the parasite in a single treatment. We also noticed reduction of parasitophorous vacuoles as an evidence of inhibition of parasite growth and resolution of infection. No substantial cytotoxicity was detected on macrophages. NONPs were able to provide a sustained parasite killing for both L. (L.) amazonensis infective stages with no toxicity on macrophages, representing a promising nanoplatform for cutaneous leishmaniasis.

TLR9/MyD88/TRIF signaling activates host immune inhibitory CD200 in Leishmania infection.

Virulent protozoans named Leishmania in tropical and subtropical areas produce devastating diseases by exploiting host immune responses. Amastigotes of Leishmania amazonensis stimulate macrophages to express CD200, an immunomodulatory ligand, which binds to its cognate receptor (CD200R) and inhibits the inducible nitric oxide synthase and nitric oxide (iNOS/NO) signaling pathways, thereby promoting intracellular survival. However, the mechanisms underlying CD200 induction in macrophages remain largely unknown. Here, we show that phagocytosis-mediated internalization of L. amazonensis amastigotes following activation of endosomal TLR9/MyD88/TRIF signaling is critical for inducing CD200 in infected macrophages. We also demonstrate that Leishmania microvesicles containing DNA fragments activate TLR9-dependent CD200 expression, which inhibits the iNOS/NO pathway and modulates the course of L. amazonensis infection in vivo. These findings demonstrate that Leishmania exploits TLR-signaling pathways not only to inhibit macrophage microbicidal function, but also to evade host systemic immune responses, which has many implications in the severity of the disease.

Amoebicidal activity of phytosynthesized silver nanoparticles and their in vitro cytotoxicity to human cells.

Acanthamoeba causes infections in humans and other animals and it is important to develop treatment therapies. Jatropha curcas, Jatropha gossypifolia and Euphorbia milii plant extracts synthesized stable silver nanoparticles (AgNPs) that were relatively stable. Amoebicidal activity of J. gossypifolia, J. curcas and E. milii leaf extracts showed little effect on viability of Acanthamoeba castellanii trophozoites. Plant-synthesized AgNPs showed higher amoebicidal activity. AgNPs synthesized by J. gossypifolia extract were able to kill 74-27% of the trophozoites at concentrations of 25-1.56 µg mL(-1) . AgNPs were nontoxic at minimum inhibitory concentration with peripheral blood mononuclear cells. These results suggest biologically synthesized nanoparticles as an alternative candidate for treatment of Acanthamoeba infections.