New morphological data of Litomosoides chagasfilhoi (Nematoda: Filarioidea) parasitizing Nectomys squamipes in Rio de Janeiro, Brazil.

Litomosoides chagasfilhoi, originally described by Moraes Neto, Lanfredi & De Souza (1997) parasitizing the abdominal cavity of the wild rodent, Akodon cursor (Winge, 1887), was found in the abdominal cavity of Nectomys squamipes (Brants, 1827), from the municipality of Rio Bonito, Rio de Janeiro State, Brazil. This study led to addition of new morphological data and a new geographical distribution for this filarioid in Brazil. Several characters were detailed and emended to previous records of L. chagasfilhoi in N. squamipes, and confirming the original description in A. cursor: buccal capsule longer than wide with walls thinner than the lumen, right spicule slightly sclerotized, with membranous distal extremity slender, with a small tongue-like terminal portion, left spicule with handle longer than the blade, whose edges form large membranous wings folded longitudinally.

New morphological data of Litomosoides chagasfilhoi (Nematoda: Filarioidea) parasitizing Nectomys squamipes in Rio de Janeiro, Brazil / Novos dados morfológicos de Litomosoides chagasfilhoi (Nematoda: Filarioidea): parasitando Nectomys squamipes no Rio de Janeiro, Brasil

Abstract Litomosoides chagasfilhoi, originally described by Moraes Neto, Lanfredi & De Souza (1997) parasitizing the abdominal cavity of the wild rodent, Akodon cursor (Winge, 1887), was found in the abdominal cavity of Nectomys squamipes (Brants, 1827), from the municipality of Rio Bonito, Rio de Janeiro State, Brazil. This study led to addition of new morphological data and a new geographical distribution for this filarioid in Brazil. Several characters were detailed and emended to previous records of L. chagasfilhoi in N. squamipes, and confirming the original description in A. cursor: buccal capsule longer than wide with walls thinner than the lumen, right spicule slightly sclerotized, with membranous distal extremity slender, with a small tongue-like terminal portion, left spicule with handle longer than the blade, whose edges form large membranous wings folded longitudinally.

Resumo Litomosoides chagasfilhoi, originalmente descrito por Moraes Neto, Lanfredi & De Souza (1997) parasitando a cavidade abdominal do roedor silvestre Akodon cursor (Winge, 1887), foi encontrado na cavidade abdominal de Nectomys squamipes (Brants, 1827), no município de Rio Bonito, Estado do Rio de Janeiro, Brasil. Este estudo propiciou a adição de novos dados morfológicos e uma nova distribuição geográfica deste filarídeo no Brasil. Vários caracteres foram detalhados e adicionados ao registro anterior de L. chagasfilhoi em N. squamipes, e confirmando a descrição original em A. cursor: cápsula bucal mais alta do que larga com paredes mais finas que o lúmen, espículo direito ligeiramente esclerotizado, com extremidade distal membranosa mais estreita, com uma pequena porção terminal em forma de língua, espículo esquerdo com cabo mais longo do que a lâmina, cujas bordas formam grandes asas membranosas dobradas longitudinalmente.

Ultrastructural alterations in adult Schistosoma mansoni, harbored in non-antihelminthic treated and low-inflammatory mice by transmission electron microscopy (TEM).

This original study suggests that alterations observed on tegumental structure and egg quality of adult Schistosoma mansoni harvested from TS mice are due to their high immune tolerogenic and low-inflammatory capacity. The tegument of worms harvested from genetically selected mice for extreme phenotypes of immune oral tolerance, resistance (TR) and susceptibility (TS) were analyzed by transmission electron microscopy (TEM). Parasites recovered from TR mice showed no tegumental morphological changes. However, specimens collected from TS mice exhibited tubercle swelling with blunted and shortened spines in lower density. These tegumental alterations were similar to those described with artemether or praziquantel treatment, but without to affecting the worm surveillance, supporting observations that the host immune system influences the development and function of the tegument of worms harbored in non-antihelminthic treated TS mice. TS mice showed a higher percentage of dead eggs and a lower percentage of immature eggs than TR mice, but had similar quantities of collected eggs. This suggests that in TS mice the alterations in adult worm tegument prevented egg development, but not egg production or worm survival. These results corroborate our previous scanning electron microscopy (SEM) study indicating the influence of the host immune regulatory profile on the development and function of the worm's reproductive system and tegument.

Synthesis, characterization and antibacterial activity of FeIII, CoII, CuII and ZnII complexes probed by transmission electron microscopy.

We synthesized iron(III), cobalt(II), copper(II) and zinc(II) complexes [Fe(III)(HBPClNOL)Cl(2)]·H(2)O (1), [Co(II)(H(2)BPClNOL)Cl(2)] (2), [Cu(II)(H(2)BPClNOL)Cl]Cl·H(2)O (3), and [Zn(II)(HBPClNOL)Cl] (4), where H(2)BPClNOL is the ligand (N-(2-hydroxybenzyl)-N-(2-pyridylmethyl)[(3-chloro)(2-hydroxy)]propylamine). The complexes obtained were characterized by elemental analysis, IR and UV-visible spectroscopies, electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS/MS), and cyclic voltammetry. X-ray diffraction studies were performed for complexes (3) and (4) revealing the presence of mononuclear and dinuclear structures in solid state for (3). However, the zinc complex is mononuclear in solid state. Biological studies of complexes (1)-(4) were carried out in vitro for antimicrobial activity against nine Gram-positive bacteria (Staphylococcus aureus strains RN 6390B, COL, ATCC 25923, Smith Diffuse, Wood 46, enterotoxigenic S. aureus FRI-100 (SEA+), FRI S-6 (SEB+) and SEC FRI-361) and animal strain S. aureus LSA 88 (SEC/SED/TSST-1+). The following sequence of inhibition promoted by the complexes was observed: (4)>(2)>(3)>(1), showing the effect of the metal on the biological activity. To directly observe the morphological changes of the internal structure of bacterial cells after the treatment, transmission electron microscopy (TEM) was employed. For the most active complex [Zn(II)(HBPClNOL)Cl] (4), granulation deposits around the genetic material and internal material leaking were clearly detected.

Detecção de fibronectina e laminina em cistos teciduais de toxoplasma gondii: ensaios imunocitoquímicos / Detection of fibronectin and laminin in toxoplasma gondii tissue cysts: immunocytochemical assays

Aims: To analyze the existence and distribution of some matrix proteins in tissue cysts of Toxoplasma gondii. Methods: Laminin and fibronectin in tissue cysts of Toxoplasma gondii were detected by confocal microscopy and transmission electron microscopy. Results: Ultrastructural immunocytochemistry showed both glycoproteins in the granular region of tissue cysts, cystic matrix, micronemes, rhoptries, dense granules and rarely at the membrane of bradyzoites of Toxoplasma gondii. Conclusions: The presence of both laminin and fibronectin in secretory organelles and in the apical region of bradyzoites suggests that exocytosis of these glycoproteins can contribute to their interaction with host cells, besides composing the cyst matrix of Toxoplasma gondii.

Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro.

Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs) as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs) as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.

Primary culture of skeletal muscle cells as a model for studies of Toxoplasma gondii cystogenesis.

Toxoplasma gondii is a protozoan pathogen of birds and mammals, including humans. The infective stage, the bradyzoite, lives within cysts, which occur predominantly in cells of the central nervous system and skeletal and cardiac muscles, characterizing the chronic phase of toxoplasmosis. In the present study, we employed for the first time primary mouse culture of skeletal muscle cells (SkMC) infected with bradyzoites, as a cellular model for cystogenesis. The interconversion of bradyzoite and tachyzoite was analyzed by immunofluorescence using 2 stage-specific antibodies, i.e., anti-bradyzoite (anti-BAG1) and anti-tachyzoite (anti-SAG1). After 24 hr of interaction only bradyzoites were multiplying, as revealed by anti-BAG1 incubation; interconversion to tachyzoites was not observed. After 48 hr of infection, 2 types of vacuoles were seen, i.e., BAG1+ and SAG1+, indicating the presence of bradyzoites as well as their interconversion to tachyzoites. After 96 hr of infection, BAG1+ vacuoles presented a higher number of parasites when compared to 48 hr, indicating multiplication of bradyzoites without interconversion. Using ultrastructural analysis, bradyzoites were found to adhere to the cell membranes via both the apical and posterior regions or were associated with SkMC membrane expansions. During bradyzoite invasion of SkMC, migration of the rough endoplasmic reticulum (RER) profiles to the parasite invasion site was observed. Later, RER profiles were localized between the mitochondria and parasitophorous vacuole membrane (PVM) that contained the parasite. After 31 days of parasite-host cell infection, RER profiles and mitochondria were not observed in association with the cyst wall. Alterations of the PVM, including increased thickness and electrondensity gain on its inner membrane face, were observed 48 hr after infection. Cystogenesis was complete 96 hr after infection, resulting in the formation of the cyst wall, which displayed numerous membrane invaginations. In addition, an electron-dense granular region enriched with vesicles and tubules was present, as well as numerous intracystic bradyzoites. These results show that the in vitro T. gondii model and SkMC are potential tools for both the study of cystogenesis using molecular approaches and the drug screening action on tissue cysts and bradyzoites.

Anionic sites on Toxoplasma gondii tissue cyst wall: expression, uptake and characterization.

Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide, which causes widespread human and animal diseases. The need for new therapeutic agents along with the biology of these parasites has fueled a keen interest in the understanding of the nutrients acquisition by these parasites. Studies on the characterization of the T. gondii cyst wall as well as the contribution of the host cell to this formation have been little explored. The aim of this paper was to investigate the electric surface charge of the T. gondii tissue cysts by ultrastructural cytochemistry, through polycationic markers, employing ruthenium red (RR) and cationized ferritin (CF). Glycosaminoglycans revealed by RR were localized on the cyst wall as a homogeneous granular layer electrondense, all over its surface. The incubation of living tissue cysts with CF for 20 min at 4 degrees C followed by the increase of temperature to 37 degrees C indicated that T. gondii cyst wall is negatively charged and that occurs an incorporation of anionic sites by the cyst wall, through vesicles and tubules, and their posterior location in the cyst matrix. So, as to identify which group of molecules produces negative charge in the cyst wall, we used enzymes for cleavage on different types of molecules, demonstrating that the negative charge in the cyst wall is mainly produced by phospholipids. Our results, described in this work show, for the first time, the negativities of the cyst wall, the incorporation and the traffic of intracellular surface molecules by T. gondii cyst wall. Our model of study can give an important contribution to the knowledge of the biology and the processes involved in nutrients acquisition by bradyzoites living inside the cysts and, and also be applied as a target for the direct action of drugs against the cyst.

Toxoplasma gondii: aspectos estruturais e funcionais da parede de cistos teciduais e cistogênese em células musculares esqueléticas, in vitro / Toxoplasma gondii: structural and functional aspects of the wall of tissue cysts and cystogenesis in skeletal muscle cells, in vitro

Toxoplasmose é uma importante doença parasitária de distribuição mundial, causada pelo Toxoplasma gondii. A necessidade de novos agentes terapêuticos contra a toxoplasmose tem despertado o interesse: (i) pela melhor caracterização da superfície celular das diferentes formas evolutivas do parasito, e (ii) por um melhor entendimento sobre os mecanismos de aquisição de nutrientes por esse patógeno assim como (iii) dos eventos relacionados à sua conversão para bradizoítos e formação do cisto tecidual. Assim, nosso primeiro objetivo foi investigar a carga elétrica de superfície dos cistos de T. gondii, através de marcadores policatiônicos, tais como o vermelho de rutênio (VR) e a ferritina cationizada (FC). Através do uso do VR foi possível identificar a localização de glicosaminoglicanos por toda a superfície da parede cística, como uma camada homogênea granular eletrondensa. A incubação de cistos vivos por 20 min a 4°C com FC confirmou que a sua superfície é carregada negativamente, havendo a subseqüente incorporação destes sítios aniônicos após a elevação da temperatura para 37°C. Nestes ensaios, inicialmente, os sítios aniônicos estavam presentes em vesículas e túbulos, e posteriormente localizados na matriz cística, próximos elou em contato direto com a membrana de bradizoítos presentes no interior dos cistos. A seguir, como o intercâmbio molecular entre os parasitos encistados e o citoplasma da célula hospedeira infectada é ainda pouco conhecido, nosso segundo objetivo foi avaliar a incorporação de traçadores endocíticos de fase fluída pelos cistos de T. gondii.

A microscopia de varredura confocal a laser (MVCL) utilizando reconstrução 3D de cistos isolados e incubados com albumina bovina (BSA) conjugada a FITC mostrou a associação do ligante à parede cística, assim como sua localização na matriz dos cistos. A atividade endocítica foi adicionalmente avaliada por estudos ultraestruturais utilizando-se diferentes traçadores (ferritina nativa, peroxidase e BSA) conjugados a partículas de ouro coloidal. A análise por microscopia eletrônica de transmissão (MET) confirmou a presença dos ligantes na superfície do cisto tecidual, dentro de vesículas e em túbulos dispersos na sua matriz, bem como associados à membrana dos bradizoítos intracísticos. Esses resultados sugerem que, pelo menos uma das vias relacionadas à aquisição de nutrientes pelos parasitos intracísticos , envolva a incorporação de moléculas disponíveis no citoplasma da célula hospedeira. Na última etapa da presente tese nosso objetivo foi acompanhar a cistogênese do T. gondii in vitro, durante a interação de bradizoítos com culturas primárias de células musculares esqueléticas (CME). A conversão de bradizoíto para taquizoíto foi monitorada entre 12-168 h de interação através de ensaios de imunofluorescência, utilizando-se anticorpos monoclonais estágio-específicos, anti-BAG1 e anti-SAG1, respectivamente.

Nossos resultados revelaram que até 24 h de interação, não houve conversão bradizoíto-taquizoíto, e que somente a partir de 48 h, vacúolos parasitóforos (VPs) positivos para SAG1 ou BAG1 puderam ser identificados numa mesma célula, sendo que os primeiros sempre continham um maior número de parasitas. Adicionalmente, a análise da interação bradizoítos-CME por microscopia eletrônica de varredura revelou a associação do parasito à célula hospedeira pela região anterior do parasito assim como, pela sua porção posterior e lateral havendo inclusive, expansão da membrana das CME. Estes dados sugerem que a invasão deste tipo celular por bradizoítos ocorra tanto por penetração ativa como por endocitose. Por fim, a análise por MET mostrou a participação de elementos do retículo endoplasmático rugoso das CME na formação do VP e permitiu evidenciar a completa cistogênese do T. gondii a partir de 96 h de infecção. Os presentes resultados fornecem importantes informações relacionadas à natureza da parede cística e sua capacidade endocítica, além de apresentar dados inéditos sobre o processo de cistogênese do T. gondii em CME. Este conjunto de informações pode efetivamente contribuir para a formulação de novas abordagens terapêuticas para toxoplasmose crônica além de revelar que o cultivo primário de CME representa uma excelente ferramenta para o estudo dos fatores envolvidos na interconversão do T. gondii.

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts.

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.