In vitro and in vivo activities of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol against Trypanosoma cruzi.

From a series of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol screened in vitro against Trypanosoma cruzi, eight (S1 to S8) were selected for in vivo screening by single-dose oral administration (200 mg/kg of body weight) to infected mice at 5 days postinfection (dpi). Based on significant decreases in both parasitemia levels and mortality rates, S2 and S3 were selected for further assays. Despite having no in vivo effect, S1 was included since it was 2-fold more potent against trypomastigotes than megazol in vitro. Trypomastigotes treated with S1, S2, or S3 showed alterations of the flagellar structure and of the nuclear envelope. When assayed on intracellular amastigotes, the selectivity index (SI) for macrophages was in the range of >27 to >63 and for cardiac cells was >32 for S1 and >48 for megazol. In noninfected mice, S1 did not alter the levels of glutamic oxalacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), or urea. S2 led to an increase in GOT, S3 to increases in GOT and GPT, and megazol to an increase in GOT. Infected mice were treated with each derivative at 50 and 100 mg/kg from dpi 6 to 15: S1 did not interfere with the course of infection or reduce the number of inflammatory foci in the cardiac tissue, S2 led to a significant decrease of parasitemia, and S3 decreased mortality. There was no direct correlation between the in vitro effect on trypomastigotes and amastigotes and the results of the treatment in experimental models, as S1 showed a high potency in vitro while, in two different schemes of in vivo treatment, no decrease of parasitemia or mortality was observed.

Serum and aqueous humour cytokine response and histopathological alterations during ocular Toxoplasma gondii infection in C57BL/6 mice.

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects most species of warm-blooded animals, and in humans it causes toxoplasmosis. Healthy people that become infected rarely present clinical symptoms because the immune system prevents the parasite from causing illness. Congenital toxoplasmosis may result in abortion, hydrocephalus, as well as neurological and ocular disease (most frequently retinochoroiditis) of the newborn. In immunocompromised patients, reactivation of latent disease can cause encephalitis. Cell-mediated immunity to T. gondii antigens involves innate acute inflammatory responses and antigen-specific adaptive immunity. Considering the complexity of the immunological events triggered during toxoplasmosis, systemic and local responses were evaluated by cytokine measurements. Aqueous humour and serum were obtained from non-infected and T. gondii Me-49 strain infected C57BL/6 mice for cytokine quantification. Histopathological analyses were made with eyes enucleated from mice after 30 days of infection. ELISA assays showed an increase of IFN-gamma levels both in serum and aqueous humour of infected mice in opposition to a decrease in IL-10 levels. On the other hand, TGF-beta was high, whereas IL-12 and TNF-alpha were present in small levels in both groups. We also detected higher levels of IL-4 and IL-6 in aqueous humour than in serum of infected mice when compared to the control group. MIP-2 presented no significant differences between the two groups. Fas and Fas-L were also present in similar levels in serum of non-infected and infected mice, but both chemokines were increased in the aqueous humour of infected mice. Histopathological analysis of infected mice showed inflammatory infiltrates around blood vessels and alteration of the outer photoreceptor segments, on the external and inner nuclear layer. Parasites were observed in 82% of eyes, inside the blood vessels associated with inflammatory infiltrate. Edema, characterized by the increase of interstitial spaces between the FTR, forming lacunae was also noted. These alterations take the form of projections (retino-vitreal), characteristic of retinochoroiditis. In conclusion, T. gondii infection of C57BL/6 mice revealed that cytokine patterns alone do not assure susceptibility or resistance against infection, thus reinforcing the notion that it is necessary more than cytokine dosage to determine Th1 or Th2 profile in this model.

Redescription and surface ultrastructure of Pygidiopsis macrostomum (Digenea: Heterophyidae).

Pygidiopsis macrostomum Travassos, 1928, a poorly known species originally described from a single specimen from Rattus norvegicus (Erxleben, 1777) in Brazil, is redescribed on the basis of metacercariae from the mesenteries of naturally infected guppies Poecilia vivipara Bloch and Schneider, 1801 (Poeciliidae), and adults obtained from an experimental infection of hamsters. Pygidiopsis macrostomum is characterized by the absence of oral spines, vitellaria extending forward to ventral sucker, uterus reaching pharyngeal level, X-shaped excretory vesicle, and an oral sucker/acetabulum ratio of 1:0.8. The surface ultrastructure shows that the tegument of the metacercaria does not strongly differ from that of adults. The brush-shaped spines of P. macrostomum are similar to those reported for Pygidiopsis summa and Pygidiopsis ardeae, but no differences in spine shape were observed throughout the body.

Absence of vacuolar membrane involving Toxoplasma gondii during its intranuclear localization.

Tachyzoites of Toxoplasma gondii were located inside the nucleus of both skeletal muscle cells infected in vitro and peritoneal exudate cells collected from infected mouse in vivo. Ultrastructural analysis demonstrated that T. gondii invades the nucleus of host cells by the parasite apical region and with constriction of its body. We noted that the rhoptry, a secretory organelle of the parasite that is involved in the host cell invasion mechanism, was empty in the intranuclear T. gondii. The parasites were found in the nuclear matrix without evidence of the vacuolar membrane. Frequently, new parasites invaded host cell nucleus, which was already infected. The significance of this nuclear invasion could reflect an alternative route of T. gondii for its transitory survival or an escape mechanism from the host immune response during the in vivo infection (or both).

Interaction of Trypanosoma cruzi with heart muscle cells: ultrastructural and cytochemical analysis of endocytic vacuole formation and effect upon myogenesis in vitro.

The process of interaction of bloodstream trypomastigotes of three different strains of Trypanosoma cruzi with heart mouse muscle cells in primary cultures, was analyzed. Differences were found in the ability of the parasites to infect the cells. Those from the Colombiana strain were more infective than those from the Y and CL strains. Infection of the cells with parasites of the Colombiana strain, but not with those of the Y strain, interfered with the normal myogenic process. Transmission electron microscopy of thin sections of heart muscle cells kept in contact with parasites for 18 h showed that many parasites are found within membrane-bounded endocytic vacuoles. Cytochemical localization of Ca2+-Mg2+-ATPase, adenylate cyclase and anionic sites (labelled with cationized ferritin) indicate that these components of the plasma membrane are not found in the membrane which lines the endocytic vacuole.

Dinitroaniline herbicides against protozoan parasites: the case of Trypanosoma cruzi.

The drugs presently in use against Chagas disease are very toxic, inducing a great number of side effects. Alternative treatments are necessary, not only for Chagas disease but also for other diseases caused by protozoan parasites where current drugs pose toxicity problems. The plant microtubule inhibitor trifluralin has previously been tested with success against Leishmania, Trypanosoma brucei and several other protozoan parasites. Trypanosoma cruzi, the causative agent of Chagas disease, is also sensitive to the drug. This sensitivity has been correlated with the deduced amino acid sequences of alpha- and beta-tubulin of T. cruzi as compared with plant, mammal and other parasite sequences.

AIDS-related lymphoma in Brazil. Histopathology, immunophenotype, and association with Epstein-Barr virus.

The occurrence of malignant lymphoma is an increasingly important cause of morbidity and mortality in AIDS patients. The incidence of AIDS-related lymphoma in some developing countries such as Brazil is increasing as the survival of HIV infection has improved. Although there is a clear association between several types of immunodeficiency-related lymphomas and Epstein-Barr virus (EBV), the association of EBV infection in AIDS-related lymphoma in Brazil, where the incidence of AIDS is high, is unknown. Formalin-fixed, paraffin-embedded tissue from 24 cases of AIDS-related lymphoma in Brazil were analyzed for morphologic classification, immunophenotype, and EBV association using in situ hybridization studies with an EBV-EBER1 biotinylated probe. Twenty cases of AIDS-related lymphoma were classified as non-Hodgkin's lymphoma and four cases were Hodgkin's disease. Eleven non-Hodgkin's lymphomas were classified as diffuse large cell type, five cases were small non-cleaved cell, Burkitt-type, and four cases were large cell immunoblastic non-Hodgkin's lymphoma. Eighteen cases were of B-cell phenotype; one was a T-cell lymphoma, and one was classified as null. Epstein-Barr virus (EBV) was demonstrated in the majority of tumor cells of 11 of 20 (55%) of the cases non-Hodgkin's lymphomas and in 3 of 4 (75%) cases of Hodgkin's disease. AIDS-related lymphomas in Brazil are usually of large cell/immunoblastic type, but Hodgkin's disease is also seen. Both non-Hodgkin's lymphoma and Hodgkin's disease are often associated with EBV infection. The non-Hodgkin's lymphoma is predominantly of B-cell phenotype.

PIP: While there is a clear association between several types of immunodeficiency-related lymphomas and Epstein-Barr virus (EBV), the association of EBV infection in AIDS-related lymphoma in Brazil, where the incidence of AIDS is high, has remained unknown. The authors report their findings from an analysis of tissue samples from 24 cases of AIDS-related lymphoma in Brazil. The samples were analyzed for morphologic classification, immunophenotype, and EBV association. 20 cases were classified as non-Hodgkin's lymphoma, while 4 were Hodgkin's disease. 11 non-Hodgkin's lymphomas were classified as diffuse large cell type, 5 as small, non-cleaved cell, Burkitt-type, and 4 as large cell immunoblastic non-Hodgkin's lymphoma. 18 cases were of B-cell phenotype; one was a T-cell lymphoma and one was classified as null. EBV was demonstrated in the tumor cells of 11 of the 20 non-Hodgkin's lymphoma cases and in 3 of the 4 cases of non-Hodgkin's disease.

Congenital Chagas's disease in an urban population: investigation of infected twins.

In the Nordeste de Amaralina suburb of Salvador Bahia, Brazil, 47 of 285 pregnant women surveyed had complement fixing antibodies to Trypanosoma cruzi. At delivery T. cruzi was detected in one of 17 placentas from the sero-positive women. The offspring of this case were premature twins and T. cruzi was detected in the peripheral blood of each before death. At autopsy the gastro-intestinal tract and urinary bladder of both were severely affected. Immunofluorescence tests on cord sera, including the single case with T. cruzi in the placenta, were negative for IgM antibodies to T. cruzi. The mother of the infected twins and three of her living children, who were born and have resided in the city, were also infected with T. cruzi. Although the children had visited an area endemic for Chagas's disease for short periods, the mode of transmission in this family may have been transplacental. The value of the immunofluorescence test in the diagnosis of congenital Chagas's disease is discussed.

Comparative ultrastructural analysis of the antennae of Triatoma guazu and Triatoma jurbergi (Hemiptera: Reduviidae) during the nymphal stage development.

The specific concept of two triatominae species of epidemiological importance in the Mato Grosso Region (Brasil), Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998, the antenniferous tubercle and the four antennal segments of nymphs from the first to fifth instar were morphologically compared by scanning electron microscopy. The main differences observed were that the antenniferous tubercle in T. guazu did not present a smaller tubercle in the base of the larger tubercle. The first antennal segment in the fifth instar had sensilla distributed with an alternating array and the trichobothria in the first instar had half of its length reaching the third antennal segment. However, in T. jurbergi the antenniferous tubercle had two smaller tubercles in the base of the two larger tubercles. The first antennal segment in the fifth instar presents sensilla distributed in pairs, and the trichobothria in the first instar has only a small portion of the structure reaching the third antennal segment. These structures differentiated the nymphs of T. guazu and T. jurbergi.

The role of RCA-binding sites in the adhesion of trypanosoma cruzi to heart muscle cells, as revealed by electron spectroscopic imaging.

The involvement of host-cell surface membrane components during T. cruzi-heart muscle cell (HMC) interaction was investigated. We used the lectin RCA I (Ricinus communis), which binds to residues of D-galactose, conjugated with ferritin as a tool to reveal the role played by lectin-binding sites during adhesion of T. cruzi to HMC. With electron spectroscopic imaging (ESI) it was possible to observe a concentration of RCA I-ferritin particles on the surface membrane of HMC at the site of parasite attachment. This suggested that migration of galactosyl residues was occurring during the cellular recognition process, particularly since these particles were absent in the immediate vicinity of the attachment site, while being present in other regions of HMC membrane not related to the attachment sites. No region of the parasite's cell body was observed to have preferential status for the purposes of adhesion to HMC.

Biological and ultrastructural effects of the anti-microtubule agent taxol against Trypanosoma cruzi.

Microtubules play fundamental roles in eukaryotic cells and have been investigated as target for drugs. Several studies showed the potential use of anti-microtubule agents against pathogenic protozoa. Taxol has been intensively studied in Leishmania spp. and microtubules have been considered as a promising antileishmanial drug target. It has been also shown that taxol interferes with the proliferation of Trypanosoma cruzi, leading to morphological alterations and interruption of nuclear division and cytokinesis. In the present work we show that T. cruzi bloodstream trypomastigotes were much more susceptible than epimastigotes, and in both forms taxol caused severe ultrastructural damage, especially associated to changes in the shape of the parasites. In trypomastigotes, different degrees of body contortion along the longitudinal axis and a marked dilatation of the flagellar pocket were detected. Treated epimastigotes presented a decrease in the electron density of the mitochondrial matrix, absence of mitochondrial cristae and an increase in the number of lipid droplets. Bizarre multi-flagellar epimastigotes were also detected, suggesting an interruption of the cytokinesis. Taxol caused no noticeable ultrastructural alterations on sub-pellicular and flagellar microtubules of both evolutive forms of T. cruzi. As already described in the literature, such structures in trypanosomatids are very resistant to microtubule disrupters when compared to those in mammalian cells. Taxol prevented the endocytosis of albumin-gold complexes by epimastigotes, and this result could be associated to the loss of the dynamic stability of the microtubules of the cytostome.

Do microtubules around the Toxoplasma gondii-containing parasitophorous vacuole in skeletal muscle cells form a barrier for the phagolysosomal fusion?

The intracellular fate of Toxoplasma gondii was studied in primary cultures of skeletal muscle cells (SMC). The labelling of secondary lysosomes with BSA-Au particles showed no phagolysosomal fusion with the vacuole containing the parasite. After internalization of the parasites, the parasitophorous vacuole became involved by closely apposed endoplasmic reticulum (ER) and mitochondria; within 18 h of interaction, microtubules were visualized in association with the parasitophorous vacuole, suggesting that they could form a barrier for the phagolysosomal fusion.

Trypanosoma cruzi-heart muscle cell interaction: the presence of two or more trypomastigotes within a single endocytic vacuole.

Ultrastructural studies of the interaction between T. cruzi clone Dm28c and heart muscle cells (HMC) showed that in the initial phase of the cell recognition process several parasites could be found attached to a focal point on the surface of the host cell. Immediately following this phase, two or more parasites could be detected inside the same endocytic vacuole by electron spectroscopic imaging (ESI).

Image analysis of two-dimensional gel electrophoresis for comparative proteomics of transgenic and non-transgenic soybean seeds.

Considering the importance of bidimensional electrophoresis and image analysis in comparative proteomics, the parameters that influence the analysis of protein expression of transgenic and non-transgenic soybean seeds were evaluated. The loaded mass of the proteins (150-500 microg), the pH separation range (3-10 or 4-7), and manual/automatic image editing were evaluated. Additionally, after optimizing the conditions, histograms and matchings were obtained in order to accurately analyze the variations (90%) in protein expression. From this, 10 proteins displayed significant differences in expression, and eight of them were characterized and identified by mass spectrometry.

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.

Dual affinity method for plasmid DNA purification in aqueous two-phase systems.

The DNA binding fusion protein, LacI-His6-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 microg of pDNA extracted per 1 mL of pUC19 desalted lysate.