Maria de Nazareth S.L. Meirelles

Effect of the alkyl-lysophospholipids on the proliferation and differentiation of Trypanosoma cruzi.

Alkyl-lysophospholipids (ALPs), designed as potential immunomodulators, have been shown to be cytotoxic for a variety of tumour cells and are under clinical studies for cancer chemotherapy. ET-18-OCH(3), hexadecylphosphocholine and ilmofosine were assayed against the three forms of Trypanosoma cruzi. Incubation with bloodstream trypomastigotes resulted, under different experimental conditions, in higher activity of the compounds in comparison with crystal violet. The ED(50)/24 h values were 13.4+/-2.8 microM and 11. 7+/-0.6 microM for amastigotes and epimastigotes, respectively. ET-18-OCH(3) (0.3 and 0.6 microM) inhibited the differentiation of epimastigotes to trypomastigotes (Dm28C clone) in the range 40-57%. This drug (3.75-15 microM) also caused a time- and dose-dependent inhibition of the intracellular proliferation of amastigotes in heart muscle cells with ED(50) values of 14.3+/-4.2, 8.9+/-1.9 and 6. 8+/-0.4 microM, after 1, 2 and 3 days of treatment. Pre-treatment of the parasite with this drug inhibited its interiorization into the host cell. Interestingly, the intracellular differentiation of amastigotes to trypomastigotes was not hampered by the drug. The present results demonstrate the lytic effect of ALPs on the three forms of T. cruzi, as well as the inhibition of both the differentiation to the infective form and the proliferation of parasites interiorized in heart cells. Ultrastructural analysis of epimastigotes treated with the three ALPs showed extensive blebing of the flagellar membrane. As described in tumour cells, the membrane seems to be a primary target of the drugs.

Trypanosoma cruzi proliferation and differentiation are blocked by topoisomerase II inhibitors.

Bacterial topoisomerase II inhibitors (ofloxacin and its commercial derivative Tarivid, nalidixic acid, and novobiocin) were tested as blockers of Trypanosoma cruzi differentiation and proliferation. The transformation of either epimastigotes into metacyclic trypomastigotes or amastigotes into trypomastigotes was inhibited by the drugs in a dose-dependent manner. The inhibition of epimastigote differentiation was also dependent on the time of drug addition to the medium. Proliferation of T. cruzi was also blocked in a dose-dependent manner by the drugs, with the exception of novobiocin, which did not inhibit epimastigote replication and resulted in cell lysis when it was used at high concentrations. On the other hand, the transformation of amastigotes into epimastigotes in axenic culture was not inhibited; this process did not require either kinetoplast (mitochondrial) DNA replication or changes in the DNA network organization. Electron microscopy of cells treated with Tarivid (ofloxacin) showed damage to the kinetoplast, suggesting that this organelle might be the target of the drug. These results indicate that a bacterial-like topoisomerase II plays an important role in T. cruzi proliferation and differentiation. Images

The Expression of Mannose Receptors in Skin Fibroblast and Their Involvement in Leishmania (L.) amazonensis Invasion

Leishmania are protozoa that invade mononuclear phagocytes with the involvement of different ligand-receptor systems, including mannose receptors. Until now, scant data are available concerning the mechanisms that govern the infection of Leishmania in other host cell types such as fibroblasts. Our aim was to analyze the expression of mannose receptors in primary cultures of skin fibroblasts (SF) further characterizing their role during the invasion of promastigotes of Leishmania (L.) amazonensis. Both fluorescent, light, and electron microscopy assays revealed that SF have mannose receptors since they bound and internalized mannosylated ligands in addition to being positively labeled by fuc-BSA-FITC probes. D-mannose competition assays revealed the participation of mannose receptors during the parasite association with SF presenting upregulated receptor expression during the initial steps of the infection. After longer periods of Leishmania:fibroblasts contact, the modulation noted in the host mannose receptors was reverted concomitantly to the infection control, suggesting that the parasites were required for the alteration maintenance and providing evidences that the SF may display microbicidal mechanisms to control the Leishmania infection.

Differential tissue tropism of Trypanosoma cruzi strains: an in vitro study

We have previously demonstrated selection favoring the JG strain of Trypanosoma cruzi in hearts of BALB/c mice that were chronically infected with an equal mixture of the monoclonal JG strain and a clone of the Colombian strain, Col1.7G2. To evaluate whether cell invasion efficiency drives this selection, we infected primary cultures of BALB/c cardiomyocytes using these same T. cruzi populations. Contrary to expectation, Col1.7G2 parasites invaded heart cell cultures in higher numbers than JG parasites; however, intracellular multiplication of JG parasites was more efficient than that of Col1.7G2 parasites. This phenomenon was only observed for cardiomyocytes and not for cultured Vero cells. Double infections (Col1.7G2 + JG) showed similar results. Even though invasion might influence tissue selection, our data strongly suggest that intracellular development is important to determine parasite tissue tropism.

Trypanosoma cruzi alters adherens junctions in cardiomyocytes

We analyzed the distribution and expression of cadherin and beta-catenin during Trypanosoma cruzi-cardiomyocyte interaction. Confocal microscopy revealed cadherin associated with beta-catenin at the cell-cell contacts. After 24h of infection, the spatial distribution and expression of both adherens junction (AJ) proteins remained unaltered. In contrast, loss of N-cadherin-catenin complex was visualized in highly infected cardiomyocytes. Immunoblotting assays corroborated the spatial disorder, showing a 46% reduction in both N-cadherin and beta-catenin expression at later infection (72h of infection). Our data demonstrate that T. cruzi infection disturbs AJs, which can result in loss of cardiac tension and may contribute to the cardiac dysfunctions present in T. cruzi infection.

Glycosylation patterns of human alpha2-macroglobulin: analysis of lectin binding by electron microscopy

Human alpha2-macroglobulin (alpha 2M) is a 720 kDa glycoprotein that presents two ultrastructural conformations: slow (S-alpha 2M) and fast (F-alpha 2M). alpha 2M acts mainly as a proteinase scavenger, but an immunomodulatory role was also proposed. This work studies the effect of desialylation and deglycosylation on the structure patterns of alpha 2M by ultrastructural analysis of lectin-induced aggregates, which represents a new approach that had never been previously used. Transmission electron microscopy (TEM) analysis showed the loss of S-alpha 2M conformation after deglycosylation, indicating that glycosidic side-chains contribute to the molecular stability of S-alpha 2M. TEM proved to be an important tool to analyze the effect of biochemical changes on alpha 2M, yielding an objective qualitative control of its morphological state. Certain carbohydrate residues did not vary between the alpha 2M conformations, since both bound similarly ConA and WGA lectins. However, the binding of PNA and BSI-B(4) was slightly lower in F-alpha 2M than in S-alpha 2M. Among the neuraminidases used to desialylate both conformations of alpha 2M that from Arthrobacter ureafaciens was the most effective. Incubation with the lectins ConA or SNA, respectively specific for mannosyl and sialyl residues, led to dose-dependent patterns of aggregation of alpha 2M molecules, mediated by lectin binding and clearly visualized by TEM.

Trypanosoma cruzi Infection Impairs the Endocytosis of Zymosan A by Cardiomyocytes

Objective: We have previously reported that mannose receptors participate and are regulated during Trypanosoma cruzi cardiomyocyte (CM) infection. Our present aim is to characterize the endocytosis of mannosylated ligands like zymosan A (Zy) in uninfected and T. cruzi-infected CM. Methods: CM infected or not by T. cruzi wereincubated with Zy for different periods of time and their internalization was analyzed at light microscopy level. Fluorescent approaches were performed by treating Zy with concanavalin-A-TRITC and washing it exhaustively prior to incubation with CM. The cultures were further stained with phalloidin-FITC and DAPI for actin and DNA visualization, respectively. Results: CM internalized Zy particles in a time-dependent fashion. The ligand specificity was confirmed by the addition of mannan, which efficiently blocked the Zy endocytosis. Designed fluorescent approaches extended and confirmed the Zy internalization by striated cells. Infected cultures displayed impairment in Zy endocytosis, which seems to be directly related to host infection rates. Conclusions: Altogether, our results show the ability of CM to ingest large particles such as the mannosylated ligand Zy. During their infection with T. cruzi, there is a loss in Zy internalization possibly due to the negative modulation of mannose receptors.

Ultrastructural differences observed in Paracoccidioides brasiliensis yeast phase cells grown on solid and in liquid medium.

An ultrastructural study was conducted on yeast‐like Paracoccidioides brasiliensis cells grown on liquid and solid peptone—yeast extract—glucose medium. A large proportion of cells grown in liquid medium presented cytoplasmic damage compared with the cells grown on solid medium, which remained intact, suggesting that agar plays an important role in the development of this fungus.

Localization of glycoconjugates and glycogen in yeast‐like cells and protoplasts of Paracoccidioides brasiliensis

Summary. The presence and localization of storage polysaccharides and of polysaccharides as cell structure constituents of Paracoccidioides brasiliensis yeast‐like cells and protoplasts were studied using the periodic acid‐thiocarbohydrazide‐silver proteinate (PATAg) technique. Yeast‐like cells presented glycogen particles in the form of rosettes, which were mostly concentrated in regions of the cytoplasm. Cells in the budding process presented small amounts of glycogen in their matrix. The intracellular membranes and the attachment of the mother cell to the bud were clearly labelled by silver, demonstrating a large amount of glycoconjugates. The protoplasts presented a small amount of glycogen in their cytoplasm, a reduction probably due to the enzymatic treatment to which the cells were submitted.