Alterations in Taenia crassiceps cysticerci cytoskeleton induced by nitazoxanide and flubendazole.

Cysticercosis is the presence of Taenia solium larval stage in tissues such as central nervous system, skin, muscles and eye globe. The current treatment is based on albendazole and praziquantel which already present resistance reports. Therefore, the search for alternative treatments is paramount. The aim of this study was to determine the effect of flubendazole and nitazoxanide on cytoskeleton proteins from Taenia crassiceps cysticerci, an experimental model for cysticercosis. Cysticerci were cultured in RPMI supplemented medium containing nitazoxanide and/or flubendazole. 24 h after the exposure the cysticerci were processed for scanning and transmission electron microscopy and for protein analysis of the cytoskeleton. The proteins were detected through 1D electrophoresis and identified through Western Blot. Nitazoxanide exposure increased tubulin and actin quantifications in T. crassiceps cysticerci. While flubendazole alone and the drugs combinations induced an increase in α-tubulin and actin and decreased ß-tubulin quantifications in the parasite. Morphological changes such as swelling and rupture of vesicle, stiff membrane, decrease in movements were observed when the cysticerci were incubated with the different compounds. In conclusion the drugs induced significative impact in the parasite`s cytoskeleton and may be considered as alternative treatments for cysticercosis.

Cytokines secretion from human mesenchymal stem cells induced by bovine bone matrix.

BACKGROUND: Bovine bone matrix is a natural material that has been used in the treatment of bone lesions. In this study, bovine bone matrix Nukbone® (NKB) was investigated due its osteoconductive and osteoinductive properties. This biomaterial induces CBFA-1 activation and osteogenic differentiation, although the cytokines involved in these processes is still unknown. OBJECTIVE: The aim of this work was to determine the influence of NKB on the pro-osteoblastic and anti-osteoblastic cytokines secretion from human mesenchymal stem cells (hMSCs). METHODS: The hMSCs were cultured onto NKB and cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ and TNF-α were analized at 0-14 days by immunoassay. In addition, hemocompatibility of NKB and characterization of hMSCs were evaluated. RESULTS: NKB induces an increase on pro-osteoblastic cytokine secretion IL-4 and a decrease on anti-osteoblastic cytokine IL-6 secretion, at days 7 and 14 of cell culture. Interestingly, there was no statistical difference between secretion profiles of others cytokines analized. CONCLUSIONS: The up-regulation of IL-4 and down-regulation of IL-6, and the secretion profiles of other cytokines examined in this work, are findings that will contribute to the understanding of the role of NKB, and similar biomaterials, in bone homeostasis and in the osteoblastic differentiation of hMSCs.

The complexity and diversity of the actin cytoskeleton of trypanosomatids.

Trypanosomatids are a monophyletic group of parasitic flagellated protists belonging to the order Kinetoplastida. Their cytoskeleton is primarily made up of microtubules in which no actin microfilaments have been detected. Although all these parasites contain actin, it is widely thought that their actin cytoskeleton is reduced when compared to most eukaryotic organisms. However, there is increasing evidence that it is more complex than previously thought. As in other eukaryotic organisms, trypanosomatids encode for a conventional actin that is expected to form microfilament-like structures, and for members of three conserved actin-related proteins probably involved in microfilament nucleation (ARP2, ARP3) and in gene expression regulation (ARP6). In addition to these canonical proteins, also encode for an expanded set of actins and actin-like proteins that seem to be restricted to kinetoplastids. Analysis of their amino acid sequences demonstrated that, although very diverse in primary sequence when compared to actins of model organisms, modelling of their tertiary structure predicted the presence of the actin fold in all of them. Experimental characterization has been done for only a few of the trypanosomatid actins and actin-binding proteins. The most studied is the conventional actin of Leishmania donovani (LdAct), which unusually requires both ATP and Mg2+ for polymerization, unlike other conventional actins that do not require ATP. Additionally, polymerized LdAct tends to assemble in bundles rather than in single filaments. Regulation of actin polymerization depends on their interaction with actin-binding proteins. In trypanosomatids, there is a reduced but sufficient core of actin-binding proteins to promote microfilament nucleation, turnover and stabilization. There are also genes encoding for members of two families of myosin motor proteins, including one lineage-specific. Homologues to all identified actin-family proteins and actin-binding proteins of trypanosomatids are also present in Paratrypanosoma confusum (an early branching trypanosomatid) and in Bodo saltans (a closely related free-living organism belonging to the trypanosomatid sister order of Bodonida) suggesting they were all present in their common ancestor. Secondary losses of these genes may have occurred during speciation within the trypanosomatids, with salivarian trypanosomes having lost many of them and stercorarian trypanosomes retaining most.

Cytoskeletal and extracellular matrix proteins resist the burning of bones.

Due the proteins from bone remains are highly resistant to pass of time and environmental conditions, they could tell us about the events that probably happened in the past. In the forensic and physical anthropology context, burnt bone remains are one of the most common pieces of recovered evidence and, generally, they are associated with funerary practices, criminal scenes or massive catastrophic events. In the present study, bone pieces of pigs were calcined at different calcination temperatures, and proteins were searched using biochemical, immunochemical and ultrastructure visualization under these experimentally conditions. For this purpose, it was successfully developed a non-demineralizing protein extraction method from burnt bone remains and the use of specific antibodies permitted the identification of different extracellular matrix and intracellular proteins. While collagen proteins type I and IV were identified and detected under middle and high calcination temperatures (300°C and 600°C); cytoskeletal proteins as actin, tubulin and, the microtubule associated protein Tau, were found under calcination process, even up high calcination temperatures. Under ultrastructural analysis, fibrous materials with a classical disposition of collagens were observed even at high calcination temperatures of the burnt bone remains. The protein identification and characterization in burnt bones as performed in present studies, is clearly demonstrating that using specific strategies for protein characterizations it is possible to found protein biomarkers in burnt bone remains and this strategy could be useful for forensic and anthropological purposes.

Differential adhesion and fibrinolytic activity of mesenchymal stem cells from human bone marrow, placenta, and Wharton's jelly cultured in a fibrin hydrogel.

Mesenchymal stem cells isolated from different tissues should share associated markers and the capability to differentiate to mesodermal lineages. However, their behavior varies in specific microenvironments. Herein, adhesion and fibrinolytic activity of mesenchymal stem cells from placenta, bone marrow, and Wharton's jelly were evaluated in fibrin hydrogels prepared with nonpurified blood plasma and compared with two-dimensional cultures. Despite the source, mesenchymal stem cells adhered through focal adhesions positive for vinculin and integrin αV in two dimensions, while focal adhesions could not be detected in fibrin hydrogels. Moreover, some cells could not spread and stay rounded. The proportions of elongated and round phenotypes varied, with placenta mesenchymal stem cells having the lowest percentage of elongated cells (~10%). Mesenchymal stem cells degraded fibrin at distinct rates, and placenta mesenchymal stem cells had the strongest fibrinolytic activity, which was achieved principally through the plasminogen-plasmin axis. These findings might have clinical implications in tissue engineering and wound healing therapy.

In Vitro Analyses Reveal the Effect of Synthetic Cytokinin Forchlorfenuron (FCF) on a Septin-Like Protein of Taeniid Cysticerci.

Cytokinin forchlorfenuron (FCF), a synthetic cytokinin, has been used specifically for the characterization of septins. In spite of genomic evidence of their existence, nothing is known about septin filaments in taeniid cestodes. The aim of this work was to determine the presence of a septin-like protein in cysticerci of Taenia crassiceps and Taenia solium using the deduced amino acid sequence of T. solium septin 4 (SEPT4_Tsm), to design and synthesize a derived immunogenic peptide (residues 88 to 103), to prepare a specific rabbit polyclonal antibody, and to examine the effects of FCF at different concentrations and exposure times on an in vitro culture of T. crassiceps cysticerci. In vitro, FCF altered the morphology and motility of T. crassiceps cysticerci, and its effects were reversible under specific concentrations. In addition, we observed by ultrastructural observation that FCF alters the cellular subunit of the protonephridial system of cestodes, where disruption of the axoneme pattern of flame cells was observed. The rabbit polyclonal antibody prepared against the synthetic peptide recognized a major band of 41 kDa in both parasites. Our results establish the importance of SEPT4_Tsm in the dynamics and survival of taeniid cysticerci, as well as their susceptibility to FCF. This is also the first report that a septin is present in the cytoskeleton of taeniids.

¿Es la patogenia del virus sincitial respiratorio humano un factor de riesgo para el desarrollo de asma infantil? / Is the pathogenesis of the human respiratory syncytial virus a risk factor for the development of childhood asthma?

Resumen El virus sincitial respiratorio humano (VSRh) es considerado como el principal agente causal de infecciones del tracto respiratorio en niños. Su presentación clínica varía en cuanto a la gravedad: desde infecciones no complicadas de la vía aérea superior en adultos y niños sanos, hasta bronquiolitis y bronconeumonía en niños con factores de riesgo y menores de 2 años. Perteneciente a la familia Pneumoviridae y al género Orthopneumovirus, el VSRh es un virus envuelto que contiene un genoma de ácido ribonucleico (RNA) monocatenario de polaridad negativa, que codifica para 7 proteínas estructurales (G, F, SH, M, P, N y L) y 4 no estructurales (NS1, NS2, M1, M2). La presencia del virus se ha considerado como factor de riesgo para el desarrollo de asma infantil, que es una enfermedad inflamatoria de la vía aérea caracterizada por episodios recurrentes de obstrucción de la vía aérea inferior ante estímulos ambientales generalmente inocuos. El riesgo de desarrollar asma aumenta si la primoinfección sucede a edad temprana y si hay factores de riesgo como prematuridad y broncodisplasia pulmonar. En México, debido a la morbilidad y mortalidad asociada al VSRh, y como profilaxis en pacientes de alto riesgo; desde el año 2008, se recomienda el uso del biofármaco Pavilizumab. El objetivo de la presente revisión es describir los factores asociados a la patogénesis VSRh que podrían estar implicados en el desarrollo del asma infantil y, con ello, plantear que población está en riesgo. Para estos fines, se presenta un breve análisis de la biología del virus, la respuesta inmune que se induce durante la infección, así como aquellos fármacos aprobados en México para el tratamiento y profilaxis de infecciones asociadas al VRSh.

Abstract The human respiratory syncytial virus (hRSV) is the main pathogen of respiratory tract infections in children. The severity of the infection is depending of its clinical presentation that is moving from uncomplicated upper airway infections, in healthy adults and children, to bronchiolitis and bronchopneumonia that could be developed, in presence of risk factors, in children younger than 2 years. The virus belongs to the Pneumoviridae family and Orthopneumovirus genus, it is an enveloped virus with a single-stranded RNA genome of negative polarity that is codifying 7 structural proteins (G, F, SH, M, P, N and L) and four non-structural proteins (NS1, NS2, M1, M2). The viral infection has been considered as a risk factor for the development of childhood asthma, which is the most common airway inflammatory disease in children and characterized, by recurrent episodes of lower airway obstruction, by harmless environmental stimuli. The risk increases if primary infection occurs at an early age and in risk factors as prematurity and pulmonary broncho-dysplasia. Due to the morbidity and mortality associated with hRSV, since 2008 it has been approved the use of biopharmaceuticals as Palivizumab for prophylaxis in high-risk patients. In the present review, the aim is to present those factors that could be involved in the development of childhood asthma and their possible link to the presence of hRSV. In addition, it is an intention for presenting the possible facts of the risks in the potentially infected population. For a better comprehension of the virus, it is presented a briefly analysis of the viral structure, the induced immune response against the viral infection and those drugs that are approved in Mexico for the treatment and prophylaxis against hRSV.

Androgens Exert a Cysticidal Effect upon Taenia crassiceps by Disrupting Flame Cell Morphology and Function.

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.

JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes.

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.

JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

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.

Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

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.

Analysis of the expression of cytoskeletal proteins of Taenia crassiceps ORF strain cysticerci (Cestoda).

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.

Proteomic characterization of the subpellicular cytoskeleton of Toxoplasma gondii tachyzoites.

Toxoplasma, the causative agent of toxoplasmosis in animals and humans, has a subpellicular cytoskeleton that is involved in motility, cell shape and invasion. Knowledge of components of the cytoskeleton is necessary to understand the invasion mechanisms as well as for the identification of possible therapeutic targets. To date, most cytoskeletal components of Toxoplasma remain unidentified due mainly to the lack of reproducible methods for their isolation. Based on the successful isolation of the cytoskeleton, it was possible to report for the first time, the proteomic characterization of the subpellicular cytoskeleton of Toxoplasma formed by 95 cytoskeletal proteins through proteomic analysis by tandem mass spectrometry of one dimension SDS PAGE. By bioinformatic analysis of the data, proteins were classified as: 18 conventional cytoskeletal proteins; 10 inner membrane complex proteins, including 7 with alveolin repeats; 5 new proteins with alveolin like repeats; 37 proteins associated with other organelles and 25 novel proteins of unknown function. One of the alveolin like proteins not previously described in Toxoplasma named TgArticulin was partially characterized with a specific monoclonal antibody. Presence of TgArticulin was exclusively associated with the cytoskeleton fraction with a cortical distribution. Functions for the several molecules identified are proposed. BIOLOGICAL SIGNIFICANCE: This manuscript describes, for the first time, the proteome of the subpellicular cytoskeleton of Toxoplasma gondii. The importance of this study is related to the role of the cytoskeleton in the highly invasive capability of a parasite that causes abortion, blindness, and death by encephalitis in immunocompromised patients. Proteomic characterization of the cytoskeleton of T. gondii tachyzoites was possible by the development of a successful procedure for the isolation of the subpellicular cytoskeleton. Knowledge of the composition of the cytoskeleton of Toxoplasma is fundamental for the understanding of the motility and host cell invasion mechanisms, and for the future design and development of toxoplasmicidal drugs with effects against specific components of the cytoskeleton of this parasite that are absent in mammal host cells.

Immunodiagnosis of porcine cysticercosis: identification of candidate antigens through immunoproteomics.

Cysticercosis, caused by the larval stage of Taenia solium, is a zoonotic disease affecting pigs and humans that is endemic to developing countries in Latin America, Africa and South East Asia. The prevalence of infection in pigs, the intermediate host for T. solium, has been used as an indicator for monitoring disease transmission in endemic areas. However, accurate and specific diagnostic tools for porcine cysticercosis remain to be established. Using proteomic approaches and the T. solium genome sequence, seven antigens were identified as specific for porcine cysticercosis, namely, tropomyosin 2, alpha-1 tubulin, beta-tubulin 2, annexin B1, small heat-shock protein, 14-3-3 protein, and cAMP-dependent protein kinase. None of these proteins were cross-reactive when tested with sera from pigs infected with Ascaris spp., Cysticercus tenuicollis and hydatid cysts of Echinococcus spp. or with serum from a Taenia saginata-infected cow. Comparison with orthologues, indicated that the amino acid sequences of annexin B1 and cAMP-dependent protein kinase possessed highly specific regions, which might make them suitable candidates for development of a specific diagnostic assay for porcine cysticercosis.

Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane.

Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

RCB20, an experimental benzimidazole derivative, affects tubulin expression and induces gross anatomical changes in Taenia crassiceps cysticerci.

Helminth ß-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.

Analysis of the effect of a 2-(trifluoromethyl)-1H-benzimidazole derivative on Trichinella spiralis muscle larvae.

Albendazole and mebendazole are widely used in the treatment of trichinellosis; however, chemotherapy failure has been reported. In an effort to develop new anthelminthic compounds, we examined a previously synthesized 2-(trifluoromethyl)-1H-benzimidazole derivative (1) that showed good in vitro activity against Trichinella spiralis muscle larvae but low in vivo efficacy. In order to improve the solubility of compound 1, an inclusion complex with 2-hydroxypropyl-ß-cyclodextrin (1/HP-ßCD) was prepared. When 1/HP-ßCD was tested in vivo, it significantly reduced the ML burden (84%). In addition, a proteomic analysis of T. spiralis ML treated with 1 revealed significant changes in the expression levels of proteins involved in energy metabolism and the cytoskeleton of the parasite. Compound (1) also induced extensive ultrastructural changes in the cuticle, hypodermis and midgut of the parasite.

In vitro and in vivo trypanocidal activity of some benzimidazole derivatives against two strains of Trypanosoma cruzi.

The trypanocidal effect of five benzimidazole derivatives (1-5) was determined in vitro and in vivo assays against two strains of Trypanosoma cruzi (NINOA and INC5). The in vitro trypanocidal activity was evaluated by measuring the percentage of lysis of bloodstream trypomastigotes of T. cruzi. Results point to 5-chloro-1H-benzimidazole-2-thiol (1) as the best activity profile compound with a 50% lytic concentration (LC(50)) of 0.014 mM (NINOA strain) and 0.32 mM (INC5 strain). Reference drugs were nifurtimox (Nfx) and benznidazole (Bnz), which on NINOA strain displayed a LC(50)=0.60 mM and LC(50)=0.78 mM, respectively; while on INC5 strain they exhibited LC(50) values of 0.31 mM and 0.69 mM, respectively. The in vivo trypanocidal activity of 1-5 on parasitemia in a murine model acute Chagas' disease indicated that 1 and Nfx showed similar activity on INC5 strain, while 5-chloro-1-methyl-1H-benzimidazole-2-thiol (2) and its regioisomer, 6-chloro-1-methyl-1H-benzimidazole-2-thiol (3), displayed better activity than Nfx and Bnz on NINOA strain. All compounds showed low cytotoxicity against Vero cells, with selective index 38-3000 times higher to the parasite.

A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor.

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-ß-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.

Visualization and 3D reconstruction of flame cells of Taenia solium (cestoda).

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.