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Androgens have been shown to exert a cysticidal effect upon Taenia crassiceps , an experimental model of cysticercosis. To further inquire into this matter, the Taenia crassiceps model was used to evaluate the expression of several proteins after testosterone (T4) and dihydrotestosterone (DHT) in vitro treatment. Under 2-D proteomic maps, parasite extracts were resolved into approximately 130 proteins distributed in a molecular weight range of 10–250 kDa and isoelectrical point range of 3–10. The resultant proteomic pattern was analysed, and significant changes were observed in response to T4 and DHT. Based on our experience with electrophoretic patterns and proteomic maps of cytoskeletal proteins, alteration in the expression of isoforms of actin, tubulin and paramyosin and of other proteins was assessed. Considering that androgens may exert their biological activity in taeniids through the non-specific progesterone receptor membrane component (PGRMC), we harnessed bioinformatics to propose the identity of androgen-regulated proteins and establish their hypothetical physiological role in the parasites. These analyses yield a possible explanation of how androgens exert their cysticidal effects through changes in the expression of proteins involved in cytoskeletal rearrangement, dynamic vesicular traffic and transduction of intracellular signals. [ABSTRACT FROM AUTHOR]

Cardiopathy is a common, irreversible manifestation of the chronic phase of Chagas disease; however, there is controversy as to how the causes for progression from the acute to the chronic phase are defined. In this work, the presence of the parasite is correlated with the occurrence of cell infiltration and fibrosis in cardiac tissues, as well as IgG detection and disease progression in a murine model. Fifty CD1 mice were infected intraperitoneally with Trypanosoma cruzi , while 30 control were administered with saline solution. Parasitemia levels were determined, and IgG titers were quantified by ELISA. At different times, randomly selected mice were euthanized, and the heart was recovered. Cardiac tissue slides were stained with HE and Masson trichrome stain. A significant increase in parasitemia levels was observed after 15 days post-infection (dpi), with a maximum of 4.1 × 10 6 parasites on 33 dpi, ending on 43 dpi; amastigote nests were observed on 15-62 dpi. Histological analysis revealed lymphocytic infiltration and fibrotic lesions from 8 dpi until the end of the study, on 100 dpi. The presence of plasma cells in the myocardium observed on 40-60 dpi, accompanied by seropositivity to ELISA on 40-100 dpi, was regarded as the hallmark of the transition phase. Meanwhile, the chronic phase, characterized by the absence of amastigotes, presence of cell infiltration, fibrotic lesions, and seropositivity, started on 62 dpi. A strong correlation between parasitemia and the presence of amastigote nests was found ( r 2 = 0.930), while correlation between the presence of fibrosis and of amastigote nests was weak ( r 2 = 0.306), and that between fibrosis and lymphocyte infiltration on 100 dpi was strong ( r 2 = 0.899). The murine model is suitable to study Chagas disease, since it can reproduce the chronic and acute phases of the human disease. The acute phase was determined to occur on 1-60 dpi, while the chronic phase starts on 62 dpi, and fibrotic damage is a consequence of the continuous inflammatory infiltration; on the other hand, fibrosis was determined to start on the acute phase, being more apparent in the chronic phase, when Chagas disease-related cardiopathy is induced., (Copyright © 2020 De Alba-Alvarado, Bucio-Torres, Zenteno, Sampedro-Carrillo, Hernández-Lopez, Reynoso-Ducoing, Torres-Gutiérrez, Guevara-Gomez, Guerrero-Alquicira, Cabrera-Bravo and Salazar-Schettino.)

Originally an anthropozoonosis in the Americas, Chagas disease has spread from its previous borders through migration. It is caused by the protozoan Trypanosoma cruzi . Differences in disease severity have been attributed to a natural pleomorphism in T. cruzi . Several post-translational modifications (PTMs) have been studied in T. cruzi , but to date no work has focused on O-GlcNAcylation, a highly conserved monosaccharide-PTM of serine and threonine residues mainly found in nucleus, cytoplasm, and mitochondrion proteins. O-GlcNAcylation is thought to regulate protein function analogously to protein phosphorylation; indeed, crosstalk between both PTMs allows the cell to regulate its functions in response to nutrient levels and stress. Herein, we demonstrate O-GlcNAcylation in T. cruzi epimastigotes by three methods: by using specific antibodies against the modification in lysates and whole parasites, by click chemistry labeling, and by proteomics. In total, 1,271 putative O-GlcNAcylated proteins and six modification sequences were identified by mass spectrometry (data available via ProteomeXchange, ID PXD010285). Most of these proteins have structural and metabolic functions that are essential for parasite survival and evolution. Furthermore, O-GlcNAcylation pattern variations were observed by antibody detection under glucose deprivation and heat stress conditions, supporting their possible role in the adaptive response. Given the numerous biological processes in which O-GlcNAcylated proteins participate, its identification in T. cruzi proteins opens a new research field in the biology of Trypanosomatids, improve our understanding of infection processes and may allow us to identify new therapeutic targets.

Human cysticercosis caused by Taenia crassiceps is unusual; however, it is an useful experimental model for cysticercosis studies. Benzimidazole derivatives are important antihelminthic drugs widely used against helminths. A novel compound 6-chloro-5-(1-naphthyloxy) -2-(trifluoromethyl)-1H-benzimidazole (RCB20) is a benzimidazole derivative less polar and more lipophilic. The aim of this study was to detect the effect of the RCB20 on the in vitro energetic metabolism of T. crassiceps cysticerci. For this, products of the metabolism both produced and secreted/excreted (S/E) by the parasite were detected through spectrophotometry and high performance liquid chromatography after exposure to 6.5 and 13 μM of RCB20 and albendazole sulfoxide (ABZSO). There was a gradual increase in the concentrations of glucose not uptaken by parasites exposed to both concentrations RCB20 and ABZSO. There was a higher concentration of all the organic acids related to the tricarboxilic acid cycle int the parasites exposed to RCB20. The structural differences between RCB20 and ABZSO result in different targets within the parasite and in a greater induction of the energetic pathways, such as the glycolysis and the TCA cycle. RCB20 is a good candidate as a substitute for anthelminthic benzimidazoles due to a differentiated site of action with similar outcome., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Biochemical studies of benzimidazole derivatives are important to determine their mode of action and activity against parasites. The lack of antihelminthic alternatives to treat parasitic infections and albendazole resistance cases make the search for new antiparasitary drugs of utmost importance. The 6-chloro-5-(1-naphthyloxy)-2-(trifluoromethyl)-1H-benzimidazole (RCB20) is a benzimidazole derivative with promising effect. This study evaluated the effect of different concentrations of RCB20 in the alternative energetic pathway of in vitro Taenia crassiceps cysticerci. The parasites were in vitro exposed to 6.5 and 13 µM of RCB20 and albendazole sulfoxide (ABZSO). The quantification of acetate, acetoacetate, β-hydroxybutyrate, fumarate and propionate was performed by high-performance liquid chromatography. The quantification of urea, creatinine and total proteins was performed by spectrophotometry. The increase in β-hydroxybutyrate reflects the enhancement of the fatty acid oxidation in the treated groups. Volatile fatty acids secretion, acetate and propionate, was increased in the treated groups. The secretion mechanisms of the treated parasites were impaired due to organic acids increased concentrations in the cysticerci. It is possible to conclude that the metabolic effect on alternative energetic pathways is slightly increased in the parasites treated with RCB20 than the ones treated with ABZSO.

The effects of testosterone (T4) and dihydrotestosterone (DHT) on the survival of the helminth cestode parasite Taenia crassiceps, as well as their effects on actin, tubulin and myosin expression and their assembly into the excretory system of flame cells are described in this paper. In vitro evaluations on parasite viability, flow cytometry, confocal microscopy, video-microscopy of live flame cells, and docking experiments of androgens interacting with actin, tubulin, and myosin were conducted. Our results show that T4 and DHT reduce T. crassiceps viability in a dose- and time-dependent fashion, reaching 90% of mortality at the highest dose used (40 ng/ml) and time exposed (10 days) in culture. Androgen treatment does not induce differences in the specific expression pattern of actin, tubulin, and myosin isoforms as compared with control parasites. Confocal microscopy demonstrated a strong disruption of the parasite tegument, with reduced assembly, shape, and motion of flame cells. Docking experiments show that androgens are capable of affecting parasite survival and flame cell morphology by directly interacting with actin, tubulin and myosin without altering their protein expression pattern. We show that both T4 and DHT are able to bind actin, tubulin, and myosin affecting their assembly and causing parasite intoxication due to impairment of flame cell function. Live flame cell video microscopy showing a reduced motion as well changes in the shape of flame cells are also shown. In summary, T4 and DHT directly act on T. crassiceps cysticerci through altering parasite survival as well as the assembly and function of flame cells.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.

The Taenia crassiceps ORF strain is used to generate a murine model of cysticercosis, which is used for diagnosis, evaluation of drugs, and vaccination. This particular strain only exists as cysticerci, is easily maintained under in vivo and in vitro conditions, and offers an excellent model for studying the cytoskeletons of cestodes. In this study, several experimental approaches were used to determine the tissue expression of its cytoskeletal proteins. The techniques used were microscopy (video, confocal, and transmission electron), one-dimensional (1D) and two-dimensional (2D) electrophoresis, immunochemistry, and mass spectrometry. The tissue expression of actin, tubulin, and paramyosin was assessed using microscopy, and their protein isoforms were determined with 1D and 2D electrophoresis and immunochemistry. Nineteen spots were excised from a proteomic gel and identified by liquid chromatography-tandem mass spectrometry and immunochemistry. The proteins identified were classic cytoskeletal proteins, metabolic enzymes, and proteins with diverse biological functions, but mainly involved in detoxification activities. Research suggests that most noncytoskeletal proteins interact with actin or tubulin, and the results of the present study suggest that the proteins identified may be involved in supporting the dynamics and plasticity of the cytoskeleton of T. crassiceps cysticerci. These results contribute to our knowledge of the cellular biology and physiology of cestodes.

Helminth β-tubulins are the targets of benzimidazole (BZM) carbamate compounds. The specificity of the interactions between such compounds and their in vivo targets depends on the presence of specific amino acid residues in the target molecules. To discover new and effective anthelmintic drugs, we used a medicinal chemistry approach to synthesize a series of BZM derivatives that exploited the BZM moiety as a template. We have previously found that one compound, 2-(trifluoromethyl)-1H-benzimidazole (RCB20), has better in vitro and in vivo activity than albendazole sulfoxide (ABZSO). In the present study, the effect of RCB20 and ABZSO treatment on expression of Taenia crassiceps cysticerci cytoskeletal proteins such as actin, myosin II, and tubulin isoforms was examined. The effects of RCB20 and ABZSO after 11 days treatment of the parasites was evaluated by light, confocal, and electron microscopy, and by immunochemistry and immunohistochemistry. The RCB20-induced effects were more rapid than the ABZSO-induced effects on the parasites. In the RCB20-treated parasites, we observed gross-structural damage at the whole parasite level, particularly in the inner tissues and flame cells. Changes in the expression patterns of the cytoskeletal proteins, as assessed by immunohistochemistry and immunoblotting, revealed that the most important drug-induced effect on the parasites was a reduction in the expression level of tyrosinated α-tubulins. Our research findings suggest that RCB20 treatment affected posttranslational modification of parasite α-tubulin molecules, which involved removal of the α-tubulin carboxy-terminal tyrosine.

The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-β-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Flame cells are the terminal cells of protonephridial systems, which are part of the excretory systems of invertebrates. Although the knowledge of their biological role is incomplete, there is a consensus that these cells perform excretion/secretion activities. It has been suggested that the flame cells participate in the maintenance of the osmotic environment that the cestodes require to live inside their hosts. In live Platyhelminthes, by light microscopy, the cells appear beating their flames rapidly and, at the ultrastructural, the cells have a large body enclosing a tuft of cilia. Few studies have been performed to define the localization of the cytoskeletal proteins of these cells, and it is unclear how these proteins are involved in cell function., Methodology/principal Findings: Parasites of two different developmental stages of T. solium were used: cysticerci recovered from naturally infected pigs and intestinal adults obtained from immunosuppressed and experimentally infected golden hamsters. Hamsters were fed viable cysticerci to recover adult parasites after one month of infection. In the present studies focusing on flame cells of cysticerci tissues was performed. Using several methods such as video, confocal and electron microscopy, in addition to computational analysis for reconstruction and modeling, we have provided a 3D visual rendition of the cytoskeletal architecture of Taenia solium flame cells., Conclusions/significance: We consider that visual representations of cells open a new way for understanding the role of these cells in the excretory systems of Platyhelminths. After reconstruction, the observation of high resolution 3D images allowed for virtual observation of the interior composition of cells. A combination of microscopic images, computational reconstructions and 3D modeling of cells appears to be useful for inferring the cellular dynamics of the flame cell cytoskeleton.

The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T.cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Using a murine model of cysticercosis caused by the Taenia crassiceps ORF strain, we developed a fluorescent quantitative evaluation of the action of two well known anti-helminthic drugs: albendazole sulfoxide and praziquantel. The fluorescence emitted by a biotransformed CellTracker Probe known as CellTracker Green CMFDA in the vesicular fluids of cysticerci was estimated, and the results were compared with macroscopic observations of the parasites. The pharmacological EC(50) value of each drug and changes in the level of biotransformation of the fluorescent tracker caused by the drugs could be easily calculated. These drug-induced changes in biotransformation could be related to changes in the GSH/GSSG ratio of parasites. Both the cysticercosis murine model and the CMFDA biotransformation assay could be used as an in vitro screening method to evaluate potential or well known cysticidal drugs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Type II myosin, the primary component of the thick filament of muscle fibers, is organized as a dimeric high molecular weight protein, and is composed of a pair of heavy chains (MHC) and two pairs of light chains. Myosin II transforms ATP energy into mechanical force. All type II myosins are conserved proteins but they have two variable regions that are located in different places of the molecule. Myosin molecules are encoded by a multigene family and many isoforms are generated. The expression of myosins depends on the developmental stage and on the type and degree of contractile activity and tissue, therefore several myosin isoforms are found in the same organism. Here we describe the use of different techniques that allowed demonstrating the presence of isoforms of the heavy chain type II myosin of Taenia solium cysticerci (larvae) and tapeworms (adults), a cestode parasite of importance in public health in many developing countries. Myosin was purified and used in comparative proteolytic fragmentation, ATPase activity, detection of antigenic differences and electrophoretic separation. The results obtained showed biochemical and immunochemical differences among cysticerci and tapeworms, and demonstrate the presence of myosin isoforms in T. solium that are probably associated to physiological requirements of each developmental stage.

Identification, localization and partial biochemical characterization of actins expressed in the larval stage of the cestode parasite Taenia solium has been carried out. Frozen tissue sections of cysticerci, the larval stage of this parasite, were reacted with rhodamine-phalloidin, parasite actin was purified by polymerization in the presence of K(+), Mg(++) and ATP actin was analyzed by SDS-PAGE and two-dimensional gel electrophoresis, and immunoblotting of actin was performed in PVDF membranes and with commercial anti-actin monoclonal antibodies. Parasitic tissues showed different fibrous actin fluorescence patterns, which correlated with the expression of isoactins. Purified globular actin had a similar molecular mass to rabbit commercial actin (approximately 44 kDa). Actin was resolved into seven isoforms, indicating a family of actin genes.