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In this work we use a methodology to study chemotaxis of Leishmania amazonensis and Trypanossoma cruzi in real time using an Optical Tweezers sy stem. We obtained qu antitative results of the parasites forces. ©2009 Optical Soci ety of America OCIS: (350.0350) General; (350.4855) Optical tweezers or optical manipulation 1. Introduction In this work, we propose a methodology to study microorganisms chemotaxis in real time using an Optical Tweezers system. Optical Tweezers allowed real time measurements of the force vectors, strength and direction, of living parasites under chemical or other kinds of gradients. This seems to be the ideal tool to perform observations of taxis response of cells and microorganisms with high sensitiv ity to capture instantaneous responses to a given stimulus. Forces involved in the movement of unicellular parasites are very small, in the femto-pico-Newton range, about the same order of magnitude of the forces generated in an Optical Tweezers. We applied this methodology to investigate the Leishmania amazonensis (L. amazonensis ) and Trypanossoma cruzi (T. cruzi ) under distinct situations. One of the fundamental goals in paras itology is the fully understanding of para site-host cell interactions. This is still more important when it comes to non-curable diseases, like Chagas disease caused by T. cruzi and leishmaniasis caused by L. amazonensis. Chagas disease is present in 18 countries of the American continent with prevalence of 16 million cases, with 4.8–5.4 million people exhibiting clinical symptoms, an annual incidence of 700,000-800,000 new cases, and 45,000 deaths due to the cardiac form of the diseas e. At present, estimates indi cate an infection prevalence of 13 million, with 3.0–3.3 million symptomatic cases and an an nual incidence of 200,000 cases in 15 countries [1]. The L. amazonensis is responsible for another serious tropical disease that affects about 30 million people in Africa, Mid Orient and Central and South America [2]. Chemotaxis is a process where cells and microorganisms direct their movements acco rding to certain chemicals in their environment. This is important for the microorganisms, such bacterias, to find food (for example, glucose) by swimming towards the highest concentration of food molecules, or to flee from poisons (for example, phenol). In multicellular organisms, chemotaxis is critical to early (e.g . movement of sperm towards the egg during fertilization) and subsequent phases of development (e.g. migration of neurons or lymphocytes) as well as in normal function. In addition, it has been recognized that mechanisms th at allow chemotaxis in animals can be su bverted during cancer metastasis. Cell signalization is responsible for the chem otatic activity and many chemical recep tor present mostly on cell membrane are involved in this kind of taxis. In order to clarify the whole infection process it is necessa ry to understand how the parasite can detect the cells. One of fundamental steps of the T. cruzi infection on invertebrate host occurs on midgut of R. prolixus , its invertebrate host. There the parasites binds to the membrane of the mi dgut cells (perimicrovillar membrane) and epimastigote forms of T. cruzi suffer intense multiplication. Studies show that the rupture of perimicrovillar membrane interrupts the multiplication and the development of the parasite in infective forms on the invertebrate host. In the parasite and the perimicrovillar membrane interaction occurs attachment between molecules from both. Quantitative measurements of the parasite chemotaxis in presence of R. prolixus perimicrovillar membrane can elucidate some points of this interaction. For a better understanding of chemotaxis it is necessary to know not only the sense and

Beads trapped by an optical tweezers can be used as a force transducer for measuring forces of the same order of magnitude as typical forces induced by flagellar motion. We used an optical tweezers to study chemotaxis by observing the force response of a flagellated microorganism when placed in a gradient of attractive chemical substances. This report shows such observations for Leishmania amazonensis, responsible for leishmaniasis, a serious disease. We quantified the movement of this protozoan for different gradients of glucose. We were able to observe both the strength and the directionality of the force. The characterization of the chemotaxis of these parasites can help to understand the mechanics of infection and improve the treatments employed for this disease. This methodology can be used to quantitatively study the taxis of any kind of flagellated microorganisms under concentration gradients of different chemical substances, or even other types of variable gradients such as temperature and pressure.

Leishmaniasis, an endemic parasitosis that leads to chronic cutaneous, mucocutaneous or visceral lesions, is part of those diseases, which still requires improved control tools. Propolis has shown activities against different bacteria, fungi, and parasites. In this study we investigated the effect of four ethanolic extracts of typified propolis collected in different Brazilian states, on Leishmania amazonensis performing assays with promastigote forms, extracellular amastigotes, and on infected peritoneal macrophages. Ethanolic extracts of all propolis samples (BRG, BRPG, BRP-1, and BRV) were capable to reduce parasite load as monitored by the percentage of infected macrophages and the number of intracellular parasites. BRV sample called red propolis, collected in the state of Alagoas, and containing high concentration of prenylated and benzophenones compounds, was the most active extract against L. amazonensis. The anti-Leishmania effect of BRV sample was increased in a concentration and time dependent manner. BRV treatment proved to be non-toxic to macrophage cultures. Since BRV extract at the concentration of 25 microg/ml reduced the parasite load of macrophages while presented no direct toxic to promastigotes and extracellular amastigotes, it was suggested that constituents of propolis intensify the mechanism of macrophage activation leading to killing of L. amazonensis. Our results demonstrate, for the first time, that ethanolic extracts of Brazilian propolis reduce L. amazonensis infection in macrophages, and encourage further studies of this natural compound in animal models of leishmaniasis.

The displacements of a dielectric microspheres trapped by an optical tweezers (OT) can be used as a force transducer for mechanical measurements in life sciences. This system can measure forces on the 50 femto Newtons to 200 pico Newtons range, of the same order of magnitude of a typical forces induced by flagellar motion. The process in which living microorganisms search for food and run away from poison chemicals is known is chemotaxy. Optical tweezers can be used to obtain a better understanding of chemotaxy by observing the force response of the microorganism when placed in a gradient of attractors and or repelling chemicals. This report shows such observations for the protozoa Leishmania amazomenzis, responsible for the leishmaniasis, a serious tropical disease. We used a quadrant detector to monitor the movement of the protozoa for different chemicals gradient. This way we have been able to observe both the force strength and its directionality. The characterization of the chemotaxis of these parasites can help to understand the infection mechanics and improve the diagnosis and the treatments employed for this disease.

In this work we applied core-shell CdS/Cd(OH)2 quantum dots (QDs) as fluorescent labels in the Leishmania amazonensis protozoarium. The nanocrystals (8-9 nm) are obtained via colloidal synthesis in aqueous medium, with final pH=7 using sodium polyphosphate as the stabilizing agent. The surface of the particles is passivated with a cadmium hydroxide shell and the particle surface is functionalized with glutaraldehyde. The functionalized and non-functionalized particles were conjugated to Leishmania organisms in the promastigote form. The marked live organisms were visualized using confocal microscopy. The systems exhibit a differentiation of the emission color for the functionalized and non-functionalized particles suggesting different chemical interactions with the promastigote moieties. Two photon emision spectra (λ exc =795nm) were obtained for the promastigotes labeled with the functionalized QDs showing a significant spectral change compared to the original QDs suspension. These spectral changes are discussed in terms of the possible energy deactivation processes.

In the present study, we evaluated the effects of hyperbaric oxygen (HBO) exposure in both Leishmania amazonensis life stages (promastigotes and amastigotes) and on macrophage cultures infected with the parasite. HBO treatment protocols, which can be tolerated by humans and animals, induced irreversible metabolic damage and affected parasite morphology, growth and ability to transform. The observation that the antioxidant N-acetylcysteine (NAC) prevents some of these deleterious effects indicated an involvement of oxidative stress during parasite HBO exposure. In addition, HBO exposed L. amazonensis-infected macrophage cultures showed reduction of the percentage of infected cells and of the number of intracellular parasites per cell. Thus, the demonstration that HBO, a therapy used in the management of different diseases, is toxic for both L. amazonensis life stages and can alter macrophage susceptibility to the infection encourages further studies of this therapy in animal models of Leishmania infection.

In this work we present a methodology to measure force strengths and directions of living parasites with an optical tweezers setup. These measurements were used to study the parasites chemotaxis in real time. We observed behavior and measured the force of: (i) Leishmania amazonensis in the presence of two glucose gradients; (ii) Trypanosoma cruzi in the vicinity of the digestive system walls, and (iii) Trypanosoma rangeli in the vicinity of salivary glands as a function of distance. Our results clearly show a chemotactic behavior in every case. This methodology can be used to study any type of taxis, such as chemotaxis, osmotaxis, thermotaxis, phototaxis, of any kind of living microorganisms. These studies can help us to understand the microorganism sensory systems and their response function to these gradients.