Biomedical subjects

Wanderley de Souza

Publications and source records attributed to Wanderley de Souza.

At least 19 recordsLinked to original sources

PubMed2006

Strategies and results of field emission scanning electron microscopy (FE-SEM) in the study of parasitic protozoa.

Field emission scanning electron microscopy (FE-SEM) provides a range of strategies for investigating the structural organization of biological systems, varying from isolated macromolecules to tissue organization and whole organisms. This review covers some of the results so far obtained using FE-SEM observation and various protocols of sample fixation to analyze the structural organization of parasitic protozoa and their interaction with host cells. The employment of FE-SEM can be broadened through the use of gold-labeled molecules or tracers, gradual extraction by detergents, and cleavage techniques. These analyses provide significant contributions to the characterization of these organisms concerning ultrastructure, cytoskeleton, motility and intracellular behavior.

Animals↗

PubMed2006

Trypanosoma cruzi exposes phosphatidylserine as an evasion mechanism.

Chagas disease is caused by Trypanosoma cruzi and affects 18 million people in Central and South America. Here we analyzed the exposure of phosphatidylserine by the different forms of the parasite life cycle. Only the infective trypomastigotes, but not the epimastigotes or intracellular amastigotes, expose this phospholipid. This triggers a transforming growth factor beta signaling pathway, based on phosphorylated Smad 2 nuclear translocation, leading to iNOS disappearance in infected macrophages. This macrophage deactivation favors the survival of this intracellular parasite. Thus, phosphatidylserine exposure may be used by T. cruzi to evade innate immunity and be a common feature of obligate intracellular parasites that have to deal with activated macrophages.

Active Transport, Cell Nucleus↗

PubMed2006

Blood and inflammatory cells of the lungfish Lepidosiren paradoxa.

A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte.

Alkaline Phosphatase↗

PubMed2006

Reservosomes: multipurpose organelles?

Reservosomes are endocytic organelles from Trypanosoma cruzi epimastigotes that store proteins and lipids for future use. The lack of molecular markers for the compartments of this parasite makes it difficult to clarify all reservosome functions, as they present characteristics of pre-lysosomes, lysosomes and recycling compartments.

Animals↗

PubMed2006

Ultrastructural alterations induced by the neolignan dihydrobenzofuranic eupomatenoid-5 on epimastigote and amastigote forms of Trypanosoma cruzi.

Eupomatenoid-5, a compound isolated from leaves of Piper regnellii var. pallescens, showed antiprotozoal activity against the epimastigote proliferative stages and intracellular amastigote forms of Trypanosoma cruzi Y strain. Eupomatenoid-5, at 7.0 microg/ml (50% growth inhibition concentration) produced morphological changes in epimastigote forms of the parasite, such as intense cytoplasmic vacuolization, mitochondrial swelling, kinetoplast alteration, presence of myelin-like figures, and mitochondrial damage, as observed by transmission electron microscopy. In amastigote forms in LLCMK(2) cells, at 7.0-microg/ml concentration, the compound induced a decrease in the number of cells with internalized parasites and in the number of internalized parasites per LLCMK(2) cell, compared with untreated cells. Furthermore, intense cytoplasmic vacuolization and autophagic vacuoles were observed in T. cruzi intracellular amastigotes after 72 h of incubation. Scanning electron microscopy confirmed the alterations in the shape of the parasites.

Animals↗

PubMed2006

Antiproliferative activities of two novel quinuclidine inhibitors against Toxoplasma gondii tachyzoites in vitro.

OBJECTIVES: To study the antiproliferative effects of ER119884 and E5700, two quinuclidine-based inhibitors of squalene synthase (SQS), against Toxoplasma gondii tachyzoites in epithelial cells. METHODS: The antiproliferative effects of the quinuclidine derivatives, alone or in combination with epiminolanosterol or antifolates, were analysed, resulting in the construction of isobolograms. The ultrastructure of treated tachyzoites was analysed by transmission electron microscopy. RESULTS: The quinuclidine derivatives demonstrated selective anti-T. gondii activity, arresting parasite growth with IC50 values of 0.66 and 0.23 microM for ER119884 and E5700, respectively, after 24 h of interaction and 0.44 and 0.19 microM after 48 h of interaction. Both compounds induced remarkable alterations in the parasite ultrastructure, such as mitochondrial swelling and the presence of autophagosome-like structures, after 24 h of treatment. Combination of these quinuclidine derivatives with the antifolates sulfadiazine and pyrimethamine produced a synergic effect. When epiminolanosterol was combined with E5700, the effect observed was synergic, whereas the combination with ER119884 produced no interaction. CONCLUSIONS: E5700 and ER119884 demonstrated selective activity against T. gondii tachyzoites and are a possible alternative to be used in association with the current therapy. The ultrastructural alterations observed suggest a possible interference with lipid metabolism.

Animals↗

PubMed2006

Secretory organelles of pathogenic protozoa.

Secretory processes play an important role on the biology and life cycles of parasitic protozoa. This review focus on basic aspects, from a cell biology perspective, of the secretion of (a) micronemes, rhoptries and dense granules in members of the Apicomplexa group, where these organelles are involved in the process of protozoan penetration into the host cell, survival within the parasitophorous vacuole and subsequent egress from the host cell, (b) the Maurer's cleft in Plasmodium, a structure involved in the secretion of proteins synthesized by the intravacuolar parasite and transported through vesicles to the erythrocyte surface, (c) the secretion of macromolecules into the flagellar pocket of trypanosomatids, and (d) the secretion of proteins which make the cyst wall of Giardia and Entamoeba, with the formation of encystation vesicles.

Animals↗

PubMed2006

An endosymbiont positively modulates ornithine decarboxylase in host trypanosomatids.

Some trypanosomatids, such as Crithidia deanei, are endosymbiont-containing species. Aposymbiotic strains are obtained after antibiotic treatment, revealing interesting aspects of this symbiotic association. Ornithine decarboxylase (ODC) promotes polyamine biosynthesis and contributes to cell proliferation. Here, we show that ODC activity is higher in endosymbiont-bearing trypanosomatids than in aposymbiotic cells, but isolated endosymbionts did not display this enzyme activity. Intriguingly, expressed levels of ODC were similar in both strains, suggesting that ODC is positively modulated in endosymbiont-bearing cells. When the aposymbiotic strain was grown in conditioned medium, obtained after cultivation of the endosymbiont-bearing strain, cellular proliferation as well as ODC activity and localization were similar to that observed in the endosymbiont-containing trypanosomatids. Furthermore, dialyzed-heated medium and trypsin treatment reduced ODC activity of the aposymbiont strain. Taken together, these data indicate that the endosymbiont can enhance the protozoan ODC activity by providing factors of protein nature, which increase the host polyamine metabolism.

Animals↗

PubMed2006

Ultrastructural study of the gametocytes and merogonic stages of Fallisia audaciosa (Haemosporina: Garniidae) that infect neutrophils of the lizard Plica umbra (Reptilia: Iguanidae).

Little is known regarding the ultrastructure of the genus Fallisia (Apicomplexa: Haemosporina: Garniidae). This report describes the fine structure of some developmental stages of Fallisia audaciosa that infect neutrophils in the peripheral blood of the Amazonian lizard Plica umbra (Reptilia: Iguanidae). The parasites lie within a parasitophorous vacuole and exhibit the basic structures of members of the Apicomplexa, such as the pellicle and the cytostome. Invaginations of the inner membrane complex were seen in the gametocytes and may be concerned with nutrition. The meronts were irregularly shaped before division, a feature unusual among members of the Apicomplexa. The unusual presence of a parasitic protozoan within neutrophils, in some way interfering with or modulating the microbicidal activity of such cells, is discussed.

Animals↗

PubMed2006

Basic biology of Pneumocystis carinii: a mini review.

Basic aspects of cell biology of Pneumocystis carinii are reviewed with major emphasis on its life cycle and the structural organization of the trophozoites and cyst forms. Initially considered as a protozoan it is now established that Pneumocystis belongs to the Fungi Kingdom. Its life cycle includes two basic forms: (a) trophozoites, which are haploid cells that divide by binary fission and may conjugate with each other forming an early procyst and (b) cysts where division takes place through a meiotic process with the formation of eight nuclei followed by cytoplasmic delimitation and formation of intracystic bodies which are subsequently released and transformed into trophozoites. Basic aspects of the structure of the two developmental stages of P. carinii are reviewed.

Animals↗

PubMed2006

Effects of sterol biosynthesis inhibitors on endosymbiont-bearing trypanosomatids.

Some protozoa of the Trypanosomatidae family have a close relationship with an endosymbiotic bacterium. As the prokaryote envelope has a controversial origin, a sterol 24-methyltransferase inhibitor (20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol; 22,26-azasterol) was used as a tool to investigate lipid biosynthetic pathways in Crithidia deanei, an endosymbiont-bearing trypanosomatid. Apart from antiproliferative effects, this drug induced ultrastructural alterations, consisting of myelin-like figures in the cytoplasm and endosymbiont envelope vesiculation. Concurrently, a dramatic reduction of 24-alkyl sterols was observed after 22,26-azasterol treatment, both in whole cell homogenates, as well as in isolated mitochondria. These effects were associated with changes of phospholipid composition, in particular a reduction of the phosphatidylcholine content and a concomitant increase in phosphatidylethanolamine levels. Lipid analyses of purified endosymbionts indicated a complete absence of sterols, and their phospholipid composition was different from that of mitochondria or whole protozoa, being similar to eubacteria closely associated with eukaryotes.

Animals↗

PubMed2006

Effects of protein kinase and phosphatidylinositol-3 kinase inhibitors on growth and ultrastructure of Trypanosoma cruzi.

An increasing number of protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. Some PK inhibitors display antiproliferative effects on protozoa. We tested three PK inhibitors on the growth and ultrastructure of epimastigotes of Trypanosoma cruzi and the effect of these drugs on intracellular amastigotes. They were staurosporine (serine/threonine kinase inhibitor), genistein (tyrosine kinase inhibitor), and wortmannin (phosphatidylinositol 3' (PI3) kinase inhibitor). All drugs inhibited epimastigote growth at the concentrations tested. Wortmannin inhibited parasite growth at the lowest concentrations. However, staurosporine was the most effective after 24 h treatment and genistein caused the stronger inhibition during the whole treatment (60-70% inhibition). The IC50 were: staurosporine: 6.43+/-1.28 microM; genistein: 6.54+/-1.86 microM; and wortmannin: 0.056+/-0.014 microM. These PK inhibitors had strong ultrastructural effects on the epimastigotes: abnormal chromatin condensation of the nucleus; loose flagellar membrane with the formation of blebs; incomplete cell division; autophagosomes and myelin-like figures. These drugs did not interfere with the division of intracellular amastigotes or with its differentiation to trypomastigotes. However, as trypanosomes have kinomes that contain a large set of protein kinases and phosphatases, PKs should not be disregarded as an important target for chemotherapy of Chagas disease.

Androstadienes↗

PubMed2005

The flagellar attachment zone of Trypanosoma cruzi epimastigote forms.

The flagellar attachment zone (FAZ) is an adhesion region of Trypanosoma cruzi epimastigote forms where the flagellum emerges from the flagellar pocket and remains attached to the cell body. This region shows a junctional complex which is formed by a linear series of apposed macular structures that are separated by amorphous material and clusters of intramembranous particles. Two protein groups appear to be important in the FAZ region: a membrane glycoprotein of 72kDa and several high molecular weight proteins. To gain a better understanding of the FAZ region, we compared wild-type Y strain T. cruzi epimastigotes with a mutant cell in which the 72-kDa surface glycoprotein (Gp72), involved in cell body-flagellum adhesion, had been deleted by target gene replacement. Using immunofluorescence confocal microscopy and electron microscopy techniques to analyze the FAZ region the results suggest that, in the absence of Gp72, other proteins involved in the formation of FAZ remain concentrated in the flagellar pocket region. The analysis of a 3-D reconstruction model of wild-type epimastigotes showed that the endoplasmic reticulum and mitochondrion are in intimate association with FAZ, in contrast to the null mutant cells where the endoplasmic reticulum was not visualized.

Animals↗

PubMed2005

Role for a P-type H+-ATPase in the acidification of the endocytic pathway of Trypanosoma cruzi.

Previous studies in Trypanosoma cruzi, the etiologic agent of Chagas disease, have resulted in the cloning and sequencing of a pair of tandemly linked genes (TcHA1 and TcHA2) that encode P (phospho-intermediate form)-type H+-ATPases with homology to fungal and plant proton-pumping ATPases. In the present study, we demonstrate that these pumps are present in the plasma membrane and intracellular compartments of three different stages of T. cruzi. The main intracellular compartment containing these ATPases in epimastigotes was identified as the reservosome. This identification was achieved by immunofluorescence assays and immunoelectron microscopy showing their co-localization with cruzipain, and by subcellular fractionation and detection of their activity. ATP-dependent proton transport by isolated reservosomes was sensitive to vanadate and insensitive to bafilomycin A1, which is in agreement with the localization of P-type H+-ATPases in these organelles. Analysis by confocal immunofluorescence microscopy revealed that epitope-tagged TcHA1-Ty1 and TcHA2-Ty1 gene products are localized in the reservosomes, whereas the TcHA1-Ty1 gene product is additionally present in the plasma membrane. Immunogold electron microscopy showed the presence of the H+-ATPases in other compartments of the endocytic pathway such as the cytostome and endosomal vesicles, suggesting that in contrast with most cells investigated until now, the endocytic pathway of T. cruzi is acidified by a P-type H+-ATPase.

Animals↗

PubMed2005

Antiproliferative and ultrastructural effects of BPQ-OH, a specific inhibitor of squalene synthase, on Leishmania amazonensis.

Parasites of the Leishmania genus require for the growth and viability the de novo synthesis of specific sterols as such as episterol and 5-dehydroepisterol because cholesterol, which is abundant in their mammalian hosts, does not fulfill the parasite sterol requirements. Squalene synthase catalyzes the first committed step in the sterol biosynthesis and has been studied as a possible target for the treatment of high cholesterol levels in humans. In this work we investigated the antiproliferative and ultrastructural effects induced by 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH), a specific inhibitor of squalene synthase, on promastigote and amastigote forms of Leishmania amazonensis. BPQ-OH had a potent dose-dependent growth inhibitory effect against promastigotes and amastigotes, with IC(50) values 0.85 and 0.11 microM, respectively. Ultrastructural analysis of the treated parasites revealed several changes in the morphology of promastigote forms. The main ultrastructural change was found in the plasma membrane, which showed signs of disorganization, with the concomitant formation of elaborated structures. We also observed alterations in the mitochondrion-kinetoplast complex such as mitochondrial swelling, rupture of its internal membrane and an abnormal compaction of the kinetoplast. Other alterations included the appearance of multivesicular bodies, myelin-like figures, alterations of the flagellar membrane and presence of parasites with two or more nuclei and kinetoplasts. We conclude that the BPQ-OH was a potent growth inhibitor of L. amazonensis, which led to profound changes of the parasite's ultrastructure and might be a valuable lead compound for the development of novel anti-Leishmania agents.

Animals↗

PubMed2005

Kinetics of pyrophosphate-driven proton uptake by acidocalcisomes of Leptomonas wallacei.

In this work, we show the kinetics of pyrophosphate-driven H+ uptake by acidocalcisomes in digitonin-permeabilized promastigotes of Leptomonas wallacei. The vacuolar proton pyrophosphatase activity was optimal in the pH range of 7.5-8.0, was inhibited by imidiodiphosphate, and was completely dependent on K+ and PPi. H+ was released with the addition of Ca2+, suggesting the presence of a Ca2+/H+ antiport. In addition, X-ray elemental mapping associated with energy-filtering transmission electron microscopy showed that most of the Ca, Na, Mg, P, K, Fe, and Zn were located in acidocalcisomes. L. wallacei immunolabeled with antibodies against Trypanosoma cruzi pyrophosphatase show intense fluorescence in cytoplasmatic organelles of size and distribution similar to the acidocalcisomes. Altogether, the results show that L. wallacei acidocalcisomes possess a H+-pyrophosphatase with characteristics of type I V-H+-PPase. However, we did not find any evidence, either for the presence of H+-ATPases or for Na+/H+ exchangers in these acidocalcisomes.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Animals↗