Papers by Javier Ambrosio
The Journal of antimicrobial chemotherapy, 2006
Praziquantel and albendazole are currently used for chemotherapeutic treatment of neurocysticerco... more Praziquantel and albendazole are currently used for chemotherapeutic treatment of neurocysticercosis. Albendazole has been found to be more effective than praziquantel; however, it is well known that not all patients will show a complete resolution of cysts. Searching for more effective treatments, this study was designed to evaluate the effect of the combination of praziquantel and albendazole sulphoxide in a Taenia crassiceps in vitro model as well as the kind of interaction between both drugs. In order to determine the concentration that produced 50% effect (EC50), T. crassiceps cysts were incubated in culture medium containing praziquantel (0.005-0.04 microg/mL), albendazole sulphoxide (0.021-0.16 microg/mL) or the combination of praziquantel and albendazole sulphoxide in a fixed-dose ratio (1:1). The experimental concentration (EC50Exp) of the combination was determined from the concentration-response curve constructed from the combined drug treatment. Isobolographic analyses w...
Molecular and Biochemical Parasitology, 2020
Trypanosomatids are a monophyletic group of parasitic flagellated protists belonging to the order... more 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.
Proteomics as a new tool in forensic sciences
The use of new technologies, such as proteomics, p... more Proteomics as a new tool in forensic sciences
The use of new technologies, such as proteomics, permits the efficient and reproducible analysis, identification and characterisation of peptides and proteins from different biological and non-biological matrices. This can help in the development of new biomarkers in
the forensic sciences. Protein markers are highly resistant to the passage of time and adverse environmental conditions, and could provide a broad overview of the physiological status of
the subjects. The use of protein markers substantially reduces the contamination of samples as compared to DNA, while providing quantitative and highly reliable data that are backed bydatabases for their interpretation. This work presents a review of the advances and limitationsof proteomics to establish the origin of the evidence found, the cause of death, the presenceof pathogens and conditional disease, as well as the biological age at death, the post-morteminterval, or the biogeographic origin of subjects.
The understanding of the cytoskeleton's importance in stem cells is essential for their manipulat... more The understanding of the cytoskeleton's importance in stem cells is essential for their manipulation and further clinical application. The cytoskeleton is crucial in stem cell biology and depends on physical and chemicals signals to define its structure. Additionally, cell culture conditions will be important in the proper maintenance of stemness, lineage commitment and differentiation. This review focuses on the role of the actin cytoskeleton of stem cells during differentiation, the significance of cellular morphology, signaling pathways involved in cytoskeletal rearrangement in stem cells, the mechanobiology and mechanotransduction processes implicated in the interactions of stem cells with different surfaces of biomaterials, such as the nanotopography which is a physical cue influencing the differentiation of stem cells. Also, cancer stem cells are included since it is necessary to understand the role of their mechanical properties to develop new strategies to treat cancer. In this context, to study the stem cells requires integrated disciplines, including molecular and cellular biology, chemistry, physics, immunology, but also mechanobiology. Finally, as one of the purposes of stem cells is their application in regenerative medicine, their deepest understanding is necessary in order to establish safety protocols and effective cell-based therapies.
The effects of testosterone (T 4) and dihydrotestosterone (DHT) on the survival of the hel-minth ... more The effects of testosterone (T 4) and dihydrotestosterone (DHT) on the survival of the hel-minth cestode parasite Taenia crassiceps, as well as their effects on actin, tubulin and myo-sin 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 T 4 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 T 4 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, T 4 and DHT directly act on T. crassiceps cysticerci through altering parasite survival as well as the assembly and function of flame cells.
Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence ... more Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence of cortical actin has been widely suggested, visualisation and localisation of actin filaments has not been reported. The subpellicular cytoskeleton network is a recently described structure possibly involved in the dynamic events. Using non-ionic detergent extractions, the cortical cytoskeleton network was enriched and used for the isolation and identification of actin. Actin was detected by Western blots in extracts of cytoskeleton networks, and it was localised by gold staining in the network and in both the apical end and the posterior polar ring. Actin was isolated from subpellicular cytoskeleton extracts by binding to DNase I, and it polymerised in vitro as filaments that were gold-decorated by a monoclonal anti-actin antibody. Filaments bound the subfragment 1 of heavy meromyosin, although with atypical arrangements in comparison with the arrowheads observed in muscle actin filaments. Treatment with cytochalasin D and colchicine altered the structural organisation of the subpellicular network indicating the participation of actin filaments and microtubules in the maintenance of its structure. Actin filaments and microtubules, in the subpellicular network, participate reciprocally in the maintaining of the parasite's shape and the gliding motility.
Teaching and observation of the inner components of cells are mainly based on bi-dimensional imag... more Teaching and observation of the inner components of cells are mainly based on bi-dimensional images. However, in present days, there are important experimental advances in the laboratory and computers for applying tridimensional observations and virtual immersion navigations inside of the cells. In the present work is shown, using a model of phagocytosis of red blood cells by macrophages, how microscopic images can be reconstructed, visualized, sectioned and used for performing virtual navigation inside of the cells. It is considered that this computational strategy of navigation can offer better capability for observation and comprehension of the inner components of the cells. These kind of computational observations could drive to propose visual hypothesis that cannot be obtained by simple microscopic observations.
Parasitology Research, 2014
The Taenia crassiceps ORF strain is used to gener-15 ate a murine model of cysticercosis, which i... more The Taenia crassiceps ORF strain is used to gener-15 ate a murine model of cysticercosis, which is used for diag-16 nosis, evaluation of drugs, and vaccination. This particular 17 strain only exists as cysticerci, is easily maintained under 18 in vivo and in vitro conditions, and offers an excellent model 19 for studying the cytoskeletons of cestodes. In this study, 20 several experimental approaches were used to determine the 21 tissue expression of its cytoskeletal proteins. The techniques 22 used were microscopy (video, confocal, and transmission 23 electron), one-dimensional (1D) and two-dimensional (2D) 24 electrophoresis, immunochemistry, and mass spectrometry. 25 The tissue expression of actin, tubulin, and paramyosin was 26 assessed using microscopy, and their protein isoforms were 27 determined with 1D and 2D electrophoresis and immuno-28 chemistry. Nineteen spots were excised from a proteomic gel 29 and identified by liquid chromatography-tandem mass spec-30 trometry and immunochemistry. The proteins identified were 31 classic cytoskeletal proteins, metabolic enzymes, and proteins 32 with diverse biological functions, but mainly involved in 33 detoxification activities. Research suggests that most 34 noncytoskeletal proteins interact with actin or tubulin, and 35 the results of the present study suggest that the proteins 36 identified may be involved in supporting the dynamics and 37 plasticity of the cytoskeleton of T. crassiceps cysticerci. These 38 results contribute to our knowledge of the cellular biology and 39 physiology of cestodes.
Cell Biology International, 2006
Type II myosins are highly conserved proteins, though differences have been observed among organi... more Type II myosins are highly conserved proteins, though differences have been observed among organisms, mainly in the filamentous region. Myosin isoforms have been identified in Taenia solium, a helminth parasite of public health importance in many developing countries. These isoforms are probably associated with the physiological requirements of each developmental stage of the parasite. In this paper we extend the characterization of myosin to several other Taenia species. Type II myosins were purified from the larvae (cysticerci) of Taenia solium, T. taeniaeformis and T. crassiceps and the adult stages of T. solium, T. taeniaeformis and T. saginata. Rabbit polyclonal antibodies against some of these myosins were specific at high dilutions but cross-reacted at low dilutions. ATPase activity was evaluated and kinetic values were calculated for each myosin. Homologous actinemyosin interactions increased both the affinity of myosin for ATP and the hydrolysis rate. The results indicate immunological and biochemical differences among taeniid myosins. This variability suggests that different isoforms are found not only in different taeniid species but also at different developmental stages. Further characterization of myosin isoforms should include determination of their amino acid composition. Ó
Revista de la …, 2009
La cisticercosis es la parasitosis más frecuente del sistema nervioso central. El principal facto... more La cisticercosis es la parasitosis más frecuente del sistema nervioso central. El principal factor de riesgo para adquirir neurocisticercosis (NCC) es la presencia en casa de un portador del estadio adulto de la Taenia solium, quien generalmente es asintomático, aunque se pueden identificar los proglótidos en su materia fecal. Con el propósito de identificar a dichos portadores se estudiaron pacientes con NCC que asistían al Servicio de Neurología del Hospital de Especialidades del Centro Médico Nacional Siglo XXI, así como a sus familias. También se incluyeron en el estudio los casos de teniosis del Instituto Nacional de Pediatría, así como los reportados por los Laboratorios Estatales de Salud Pública del Estado de México y de Tlaxcala. Se analizaron los cuadros clínicos de los enfermos con NCC y con teniosis. Las manifestaciones clínicas de NCC encontradas fueron similares a las descritas en la literatura. En pacientes con teniosis, la cefalea, hiperorexia, dolor en hipocondrio derecho y estreñimiento se presentaron con significancia estadística. Fue notoria la dificultad técnica para el diagnóstico de teniosis y para la obtención del parásito una vez administrado el tratamiento, así como la falta de información sobre la enfermedad, por parte de los pacientes con teniosis y sus familias.
Biochemical and Biophysical Research Communications, 2013
Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells.
Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence ... more Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence of cortical actin has been widely suggested, visualisation and localisation of actin filaments has not been reported. The subpellicular cytoskeleton network is a recently described structure possibly involved in the dynamic events. Using non-ionic detergent extractions, the cortical cytoskeleton network was enriched and used for the isolation and identification of actin. Actin was detected by Western blots in extracts of cytoskeleton networks, and it was localised by gold staining in the network and in both the apical end and the posterior polar ring. Actin was isolated from subpellicular cytoskeleton extracts by binding to DNase I, and it polymerised in vitro as filaments that were gold-decorated by a monoclonal anti-actin antibody. Filaments bound the subfragment 1 of heavy meromyosin, although with atypical arrangements in comparison with the arrowheads observed in muscle actin filaments. Treatment with cytochalasin D and colchicine altered the structural organisation of the subpellicular network indicating the participation of actin filaments and microtubules in the maintenance of its structure. Actin filaments and microtubules, in the subpellicular network, participate reciprocally in the maintaining of the parasite's shape and the gliding motility.
Journal of Visualized Experiments, 2015
Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when ... more 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.
PLOS ONE, 2015
The effects of testosterone (T4) and dihydrotestosterone (DHT) on the survival of the helminth ce... more 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.
Transactions of the Royal Society of Tropical Medicine and Hygiene
An intervention study with mass treatment against taeniasis to prevent neurocysticercosis due to ... more An intervention study with mass treatment against taeniasis to prevent neurocysticercosis due to Taenia solium in a rural community in Mexico was performed in 1991-96. Information and biological samples were obtained at the beginning of the study, at 6 months and at 42 months after mass treatment with praziquantel at a single dose of 5 mg/kg. Prevalence rates of taeniasis were measured by the detection of Taenia coproantigens and Taenia eggs in faeces; neurocysticercosis was suggested by clinical data and by serum antibodies in humans and also in swine. A reduction of 53% after 6 months and of 56% after 42 months for human taeniasis was seen after treatment. Late-onset general seizures decreased 70%. Anti-cysticercus antibodies in the human population were reduced by 75% after 42 months. Antibodies in pigs also showed a significant reduction of 55% after 6 months. In conclusion, an impact of mass chemotherapy against taeniasis to control cysticercosis in the short and long term was ...
International Journal of Morphology, 2011
No existen, hasta el momento, imágenes que muestren la disposición de la citoarquitectura de pará... more No existen, hasta el momento, imágenes que muestren la disposición de la citoarquitectura de parásitos adultos de Taenia solium, parásitos los cuales se encuentran en el intestino de portadores humanos asintomáticos. Las causas de ello podrían tener como base el que cuando se recuperan los parásitos, ellos han sufrido alteraciones debidas a la respuesta inmune de sus hospederos o bien, por el efecto que han producido en los parásitos los fármacos antihelmínticos que hayan sido usados en el tratamiento de los pacientes. Una de las alternativas que se han encontrado para la obtención de parásitos adultos, es la obtención de tenias a partir del modelo de teniosis experimental en hámsteres dorados e inmunosuprimidos y que gracias a este modelo se han podido efectuar diferentes tipos de estudios de los parásitos de esta fase infectiva. El propósito de este reporte es presentar imágenes de ultraestructura, obtenidas mediante Microscopía Electrónica de Barrido, de un corte transversal obtenido de un proglótido de una tenia recuperada de una infección experimental. Las imágenes se obtuvieron a diferentes aumentos y muestran aspectos relacionados con la superficie tegumentaria, el tegumento sincicial continuo, la capa germinal que incluye el soma de algunas células subtegumentarias y los ductos del sistema protonefridial tanto vacíos como llenos con corpúsculos calcáreos. Las imágenes ultraestructurales obtenidas muestran una forma de observación de la anatomía microscopica de los parásitos en estudio y ello contribuye a ampliar el conocimiento de los mismos en relación a aspectos de su biología celular y su fisiología.
Memórias do Instituto Oswaldo Cruz, 2014
Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of micro... more 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.
International Journal for Parasitology, 2014
We examined the effects of oestradiol (E 2 ) and progesterone (P 4 ) on cytoskeletal protein expr... more We examined the effects of oestradiol (E 2 ) and progesterone (P 4 ) 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 E 2 -and P 4 -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 E 2 and P 4 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 E 2 and P 4 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.
Steroids, 2011
The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is de... more 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--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.
PLoS ONE, 2011
Background: Flame cells are the terminal cells of protonephridial systems, which are part of the ... more 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.
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Papers by Javier Ambrosio
The use of new technologies, such as proteomics, permits the efficient and reproducible analysis, identification and characterisation of peptides and proteins from different biological and non-biological matrices. This can help in the development of new biomarkers in
the forensic sciences. Protein markers are highly resistant to the passage of time and adverse environmental conditions, and could provide a broad overview of the physiological status of
the subjects. The use of protein markers substantially reduces the contamination of samples as compared to DNA, while providing quantitative and highly reliable data that are backed bydatabases for their interpretation. This work presents a review of the advances and limitationsof proteomics to establish the origin of the evidence found, the cause of death, the presenceof pathogens and conditional disease, as well as the biological age at death, the post-morteminterval, or the biogeographic origin of subjects.
The use of new technologies, such as proteomics, permits the efficient and reproducible analysis, identification and characterisation of peptides and proteins from different biological and non-biological matrices. This can help in the development of new biomarkers in
the forensic sciences. Protein markers are highly resistant to the passage of time and adverse environmental conditions, and could provide a broad overview of the physiological status of
the subjects. The use of protein markers substantially reduces the contamination of samples as compared to DNA, while providing quantitative and highly reliable data that are backed bydatabases for their interpretation. This work presents a review of the advances and limitationsof proteomics to establish the origin of the evidence found, the cause of death, the presenceof pathogens and conditional disease, as well as the biological age at death, the post-morteminterval, or the biogeographic origin of subjects.