Renato Augusto DaMatta

Increased association of Trypanosoma cruzi with sialoadhesin positive mice macrophages.

Trypanosoma cruzi is a parasite with large amounts of sialic acid (SA) residues exposed at its surface that seems to be involved in macrophages infection. Some macrophages, present in T. cruzi infected tissues, expresses sialoadhesin (Sn), a receptor that recognizes SA. Thus, the involvement of Sn in the association of T. cruzi to macrophages was investigated. Sn was induced in mice peritoneal macrophages by homologous serum (HS) cultivation. Epimastigotes and trypomastigotes associated more to HS cultured macrophages than to fetal bovine serum (FBS). Blocking of Sn with antibodies reduced the association of trypomastigotes to similar level as for FBS cultured macrophages. Desialylation reduced the association of parasites to HS cultured macrophages indicating the Sn importance. Furthermore, the entrance mechanism of trypomastigotes to Sn positive macrophages has a phagocytic nature as demonstrated by scanning electron microscopy and cytochalasin D treatment. Sn positive macrophages may important in the initial trypomastigote infection, thus in the establishment of Chagas disease.

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 microg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.

Phosphatidylserine Exposure by Toxoplasma gondii Is Fundamental to Balance the Immune Response Granting Survival of the Parasite and of the Host

Phosphatidylserine (PS) exposure on the cell surface indicates apoptosis, but has also been related to evasion mechanisms of parasites, a concept known as apoptotic mimicry. Toxoplasma gondii mimics apoptotic cells by exposing PS, inducing secretion of TGF-beta1 by infected activated macrophages leading to degradation of inducible nitric oxide (NO) synthase, NO production inhibition and consequently persisting in these cells. Here PS+ and PS− subpopulation of tachyzoites were separated and the entrance mechanism, growth and NO inhibition in murine macrophages, and mice survival and pathology were analyzed. Infection index in resident macrophages was similar for both PS subpopulations but lower when compared to the total T. gondii population. Growth in resident macrophages was higher for the total T. gondii population, intermediate for the PS+ and lower for the PS− subpopulation. Production of NO by activated macrophages was inhibited after infection with the PS+ subpopulation and the total populations of tachyzoites. However, the PS− subpopulation was not able to inhibit NO production. PS+ subpopulation invaded macrophages by active penetration as indicated by tight-fitting vacuoles, but the PS− subpopulation entered macrophages by phagocytosis as suggested by loose-fitting vacuoles containing these tachyzoites. The entrance mechanism of both subpopulations was confirmed in a non-professional phagocytic cell line where only the PS+ tachyzoites were found inside these cells in tight-fitting vacuoles. Both subpopulations of T. gondii killed mice faster than the total population. Clear signs of inflammation and no tachyzoites were seen in the peritoneal cavity of mice infected with the PS− subpopulation. Moreover, mice infected with the PS+ subpopulation had no sign of inflammation and the parasite burden was intense. These results show that PS+ and PS− subpopulations of T. gondii are necessary for a successful toxoplasma infection indicating that both subpopulations are required to maintain the balance between inflammation and parasite growth.

ENDOGENOUS POLYAMINE LEVELS IN MACROPHAGES IS SUFFICIENT TO SUPPORT GROWTH OF TOXOPLASMA GONDII

Cytotoxic-activated macrophages control Toxoplasma gondii growth by producing nitric oxide (NO). However, the parasite can partially inhibit NO production. NO is generated from arginine within the polyamine biosynthetic pathway. Two enzymes of this pathway are ornithine decarboxylase (ODC) and arginine decarboxylase (ADC). The aim of the present work was to investigate whether T. gondii is able to modulate polyamine metabolism in macrophages. Toxoplasma gondii infection did not affect basal ODC or ADC activity. However, lipopolysaccharide induced an increase in ODC activity. Polyamine-treated macrophages exhibited a T. gondii–infection index similar to controls but a higher adhesion index; the parasite did not grow in methyl-ornithine (ODC inhibitor)–treated macrophages. The parasites were able to take up putrescine with a Km of 0.92 μM, indicating the presence of a high-affinity putrescine-transporter system. Putrescine-treated T. gondii actively penetrated macrophages and Vero cells. However, NO production and lysosomal parasitophorous vacuole fusion were not inhibited. Considered together, these results demonstrate that T. gondii requires polyamines for multiplication. However, as opposed to Trypanosoma cruzi and because of a relatively high-affinity putrescine-transporter system in the parasite, constitutive macrophage levels of putrescine seem sufficient to support T. gondii survival and multiplication.

Nitric oxide is not involved in the killing of Trypanosoma cruzi by chicken macrophages

Abstract It is known that chicken macrophages derived in vitro from blood monocytes have the capacity to destroy Trypanosoma cruzi, but Toxoplasma gondii can survive within these cells. This study was performed to determine the involvement of nitric oxide (NO) in the killing of T. cruzi by chicken macrophages. Activated (by interferon-γ and lipopolysaccharide) mouse peritoneal macrophages were used as controls. Macrophages were infected with T. cruzi and T. gondii; after 2, 24, and 48 h, NO was assayed using the Griess reagent. Respiratory-burst involvement, revealed by the reduction of nitroblue tetrazolium (NBT), was determined in chicken macrophages. Chicken macrophages did not produce NO; mouse macrophages were capable of producing NO with no multiplication of parasites. Reduction of NBT could be detected in chicken macrophages that interacted with T. cruzi but was absent in those that interacted with T. gondii. These results demonstrate that chicken macrophages do not use NO as a microbicidal agent when infected with T. cruzi or T. gondii.

A new type of pterocarpanquinone that affects Toxoplasma gondii tachyzoites in vitro

Toxoplasma gondii, the agent of Toxoplasmosis, is an obligate intracellular protozoan able to infect a wide range of vertebrate cells, including nonprofessional and professional phagocytes. Therefore, drugs must have intracellular activities in order to control this parasite. The most common therapy for Toxoplasmosis is the combination of sulfadiazine and pyrimethamine. This treatment is associated with adverse reactions, thus, the development of new drugs is necessary. In previous studies, naphthoquinone derivatives showed anti-cancer activity functioning as agents capable of acting on groups of DNA, preventing cancer cells duplication. These derivatives also display anti-parasitic activity against Plasmodium falciparum and Leishmania amazonensis. The derivative pterocarpanquinone tested in this work resulted from the molecular hybridization between pterocarpans and naphtoquinone that presents anti-tumoral and anti-parasitic activities of lapachol. The aim of this work was to determine if this derivative is able to change T. gondii growth within LLC-MK2 cells. The drug did not arrest host cell growth, but was able to decrease the infection index of T. gondii with an IC(50) of 2.5 μM. Scanning and transmission electron microscopy analysis showed morphological changes of parasites including membrane damage. The parasite that survived tended to encyst as seen by Dolichos biflorus lectin staining and Bag-1 expression. These results suggest that pterocarpanquinones are drugs potentially important for the killing and encystment of T. gondii.

l-Amino acid oxidase activity present in fractions of Bothrops jararaca venom is responsible for the induction of programmed cell death in Trypanosoma cruzi

Bothrops jararaca venom induces programmed cell death in epimastigotes of Trypanosoma cruzi. Here we fractionated the venom and observed that the anti-T. cruzi activity was associated with fractions that present L-amino acid oxidase (L-AAO) activity. L-AAO produces H(2)O(2), which is highly toxic. The addition of catalase to the medium, a H(2)O(2) scavenger, reverted the killing capacity of venom fractions. The anti-T. cruzi activity was also abolished when parasites were cultured in a medium without hydrophobic amino acids that are essential for L-AAO activity. These results were confirmed with a commercial purified L-AAO. Treatment for 24 h with fractions that present L-AAO activity induced parasites cytoplasmic retraction, mitochondrial swelling and DNA fragmentation, all morphological characteristics of programmed cell death. Similar changes were also observed when parasites were treated with H(2)O(2). These results indicate that H(2)O(2), the product of L-AAO reaction, induces programmed cell death explaining the anti-T. cruzi activity of B. jararaca venom.

Leukocyte ultrastructure, hematological and serum biochemical profiles of ostriches (Struthio camelus)

In the last decade ostrich farms spread throughout the world as an alternative source of investment. Although previous studies have reported hematology and biochemical values for ostriches from several regions of the world, little information is available regarding leukocyte morphology. This study reports the morphology and ultrastructure of ostrich leukocytes and hematology and biochemical values from birds raised in Brazil. Heterophils presented a lobulated nucleus, and fusiform, and acidophilic and peroxidase-negative granules. Ultrastructurally, 2 kinds of cytoplasmic granules were observed: one was large and fusiform and the other smaller with heterogeneous morphology and electrondensity; granules were peroxidase-negative. Eosinophils had a kidney-shaped eccentrically placed nucleus that was rarely lobulated and eosinophilic, round, and peroxidase-positive granules. At the ultrastructure level, 2 main kinds of granules with the same size and form but different electron density were seen; granules were peroxidase-positive. Lymphocytes and thrombocytes had the same characteristics of other avian species; monocytes presented morphological heterogeneity. Hematological and serum biochemical profiles had no sex influence and were established for ostriches raised in southeastern Brazil. These parameters will help the diagnosis of specific ostrich pathologies and serve as basic knowledge for studies in immunology and comparative avian pathology.

Occurrence of nematodes and anthelmintic management of ostrich farms from different Brazilian states: Libyostrongylus douglassii dominates mixed infections

Ostriches were imported to Brazil in the early 1990s without appropriate parasite control leading to a co-importation of parasites. Libyostrongylus douglassii, Libyostrongylus dentatus and Codiostomum struthionis are nematodes of the ostriches that have been reported on some Brazilian farms but no national survey has been performed. Moreover, little is known about anthelmintic usage to control these parasites in Brazil. Therefore, the occurrence of L. douglassii, L. dentatus and C. struthionis and the anthelmintic management practices in Brazilian ostrich farms were studied. A questionnaire about management practices and information on how to collect, store and ship feces to the laboratory were sent to 17 farms located in 9 Brazilian states. Received fecal samples were submitted to fecal cultivation and larval harvested. Infective larvae were morphologically analyzed and quantified. The majority of the farms (88%) used an anthelmintic at least once a year. Ivermectin was the principal anthelmintic. Libyostrongylus sp. and C. struthionis were found in samples from 76 and 18% of the examined farms, respectively. The presence of L. douglassii (61-97%) was higher than L. dentatus (3-57%) and C. struthionis (2-30%). At the farm level, both species of Libyostrongylus were always found together. Sheath tail measurements of their infective larvae were crucial for morphological diagnosis. L. dentatus and L. douglassii are well adapted to Brazil and are widely distributed in the Brazilian territory, proving that this mixed infection in Brazilian ostrichs farms is common.

Volutin Granules of Eimeria Parasites are Acidic Compartments and Have Physiological and Structural Characteristics Similar to Acidocalcisomes

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti‐parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X‐ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.