RESUMEN
A series of aminophenylhydroxamates and aminobenzylhydroxamates were synthesized and screened for their antiparasitic activity against Leishmania, Trypanosoma, and Toxoplasma. Their anti-histone deacetylase (HDAC) potency was determined. Moderate to no antileishmanial or antitrypanosomal activity was found (IC50â¯>â¯10⯵M) that contrast with the highly efficient anti-Toxoplasma activity (IC50â¯<â¯1.0⯵M) of these compounds. The antiparasitic activity of the synthetized compounds correlates well with their HDAC inhibitory activity. The best-performing compound (named 363) express a high anti-HDAC6 inhibitory activity (IC50 of 0.045⯱â¯0.015⯵M) a moderate cytotoxicity and a high anti-Toxoplasma activity in the range of known anti-Toxoplasma compounds (IC50 of 0.35-2.25⯵M). The calculated selectivity index (10-300 using different human cell lines) of the compound 363 makes it a lead compound for the future development of anti-Toxoplasma molecules.
Asunto(s)
Antiparasitarios/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/efectos de los fármacos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Antiparasitarios/síntesis química , Línea Celular , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/aislamiento & purificación , Humanos , Ácidos Hidroxámicos/química , Concentración 50 Inhibidora , Leishmania/efectos de los fármacos , Estructura Molecular , Relación Estructura-Actividad , Toxoplasma/efectos de los fármacos , Trypanosoma/efectos de los fármacosRESUMEN
Toxplasma is a protozoan parasite, which forms persistent cysts in tissues of chronically infected animals and humans. Cysts can reactivate leading to severe pathologies. They also contribute to the transmission of Toxoplasma infection in humans by ingestion of undercooked meat. Classically, the quantification of cyst burden in tissues uses microscopy methods, which are laborious and time consuming. Here, we have developed automated protocols to quantify cysts, based on flow cytometry or high-throughput microscopy. Brains of rodents infected with cysts of Prugniaud strain were incubated with the FITC-Dolichos biflorus lectin and analyzed by flow cytometry and high-throughput epifluorescence microscopy. The comparison of cyst counts by manual epifluorescence microscopy to flow cytometry or to high-throughput epifluorescence microscopy revealed a good correlation (r = 0.934, r = 0.993, P < 0.001 respectively). High-throughput epifluorescence microscopy was found to be more specific and sensitive than flow cytometry and easier to use for large series of samples. This reliable and easy protocol allow the specific detection of Toxoplasma cysts in brain, even at low concentrations; it could be a new way to detect them in water and in contaminate food.
Asunto(s)
Automatización de Laboratorios/métodos , Encéfalo/patología , Recuento de Células/métodos , Citometría de Flujo/métodos , Ensayos Analíticos de Alto Rendimiento , Toxoplasma/citología , Toxoplasmosis Animal/patología , Animales , Encéfalo/parasitología , Femenino , Fluoresceína-5-Isotiocianato/análisis , Humanos , Lectinas/análisis , Carne/parasitología , Ratones , Ratas , Ratas Endogámicas , Sensibilidad y Especificidad , Extractos de Tejidos , Toxoplasmosis Animal/parasitología , beta-Galactosidasa/análisisRESUMEN
OBJECTIVE: The hematopoietic microenvironment is complex, and the role of myofibroblast in its function is crucial. In order to obtain a stable model reflecting this particular cell type, we have previously established human bone marrow cell lines from primary myofibroblastic Stro1(+) population (pStro1(+)). We placed HPV16 E6 and E7 expression under the control of different promoters. Here, we have characterized and studied the hematopoietic support for two cell lines corresponding to the promoters alpha-SM (alphaSM-56 line) and SV40 (SV40-56 line). MATERIALS AND METHODS: The expression profile was analyzed at the RNA level by gene array and at the protein level by Western blot, flow cytometry, and ELISA. Hematopoietic support determined using colony-forming unit (CFU) and stroma-adherent colony-forming cell (SA-CFC) assays. RESULTS: The phenotype of cell lines was not significantly modified compared with primary myofibroblastic cells. They secreted a broad spectrum of hematopoietic cytokines and nonspecific mediators. The two lines allowed the growth of hematopoietic precursors and had different support capabilities. CONCLUSIONS: We have extensively characterized two novel human bone marrow stromal cell lines. They retained a myofibroblastic phenotype and have substantial but different hematopoietic support capabilities. These lines provided a basis for determining stromal factors involved in stem-cell regulation.
Asunto(s)
Citocinas/genética , Células Madre Hematopoyéticas/fisiología , Células del Estroma/fisiología , Antígenos CD34/análisis , Células de la Médula Ósea/citología , Adhesión Celular , Moléculas de Adhesión Celular/análisis , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Criopreservación , Medios de Cultivo , Citocinas/análisis , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Humanos , Integrinas/análisis , Fenotipo , Células del Estroma/citologíaRESUMEN
The in vitro study of mammalian hematopoiesis is hindered by the lack of immortalized human stromal cell lines that support hematopoiesis. We have immortalized human stromal vascular smooth muscle cells characterized by the expression of the alpha-smooth muscle (alpha-SM) actin. This marker is usually down-regulated as a result of oncogenic transformation. To correct this dedifferentiation, we placed the expression of human papilloma virus 16 E6/E7 oncogenes under the control of the tissue-specific alpha-SM actin promoter. The immortalization event is rare and requires polyclonal culture, but the corresponding established line retains alpha-SM actin expression. Moreover, when compared with other lines derived from the same cells from vectors made with the same oncogenes but driven by either an internal SV40 promoter or the viral long terminal repeat, this line is less transformed as shown by anchorage-independent growth assay. We show therefore that the use of a physiological promoter allows the production of human cell lines with a conserved phenotype.