RESUMEN
Human trophoblast research relies on a combination of in vitro models, including isolated primary cultures, explant cultures, and trophoblast cell lines. In the present study, we have utilized the rotating wall vessel (RWV) bioreactor to generate a three-dimensional (3-D) model of human placentation for the study of cytotrophoblast (CTB) invasion. The RWV supported the growth of the human CTB cell line SGHPL-4 and allowed for the formation of complex, multilayered 3-D aggregates that were morphologically, phenotypically, and functionally distinct from SGHPL-4 monolayers. The cells cultured three-dimensionally differentiated into an aggressively invasive cell population characterized by the upregulation of matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-9 and urokinase-type plasminogen activator (uPA) secretion and activation. Microarray analysis of the 3-D and 2-D cultured cells revealed increased expression in the 3-D cells of various genes that are known mediators of invasion, including MT1-MMP, PECAM-1 and L-selectin, as well as genes not previously associated with CTB differentiation such as MMP-13 and MT5-MMP. These results were verified by quantitative real-time PCR. These findings suggest that when cultured in 3-D, SGHPL-4 cells closely mimic differentiating in utero CTBs, providing a novel approach for the in vitro study of the molecular mechanisms that regulate CTB differentiation and invasion.
Asunto(s)
Placentación/fisiología , Trofoblastos/citología , Reactores Biológicos , Western Blotting , Agregación Celular/fisiología , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular , Femenino , Humanos , Selectina L/biosíntesis , Selectina L/genética , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/enzimología , Trofoblastos/metabolismo , Trofoblastos/ultraestructura , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Hair cell mechano-electric transducer (MET) channels play a pivotal role in auditory and vestibular signal detection, yet few data exist regarding their molecular nature. Present work characterizes the MET channel pore, a region whose properties are thought to be intrinsically determined. Two approaches were used. First, the channel was probed with antagonists of candidate channel subtypes including: cyclic nucleotide-gated channels, transient receptor potential channels and gap-junctional channels. Eight new antagonists were identified. Most of the effective antagonists had a partially charged amine group predicted to penetrate the channel pore, antagonizing current flow, while the remainder of the molecule prevented further permeation of the compound through the pore. This blocking mechanism was tested using curare to demonstrate the open channel nature of the block and by identifying methylene blue as a permeant channel blocker. The second approach estimated dimensions of the channel pore with simple amine compounds. The narrowest diameter of the pore was calculated as 12.5 +/- 0.8 A and the location of a binding site approximately 45% of the way through the membrane electric field was calculated. Channel length was estimated as approximately 31 A and the width of the pore mouth at < 17 A. Each effective antagonist had a minimal diameter, measured about the penetrating amine, of less than the pore diameter, with a direct correlation between IC(50) and minimal diameter. The IC(50) was also directly related to the length of the amine side chains, further validating the proposed pore blocking mechanism. Data provided by these two approaches support a hypothesis regarding channel permeation and block that incorporates molecular dimensions and ion interactions within the pore.
Asunto(s)
Células Ciliadas Auditivas Internas/fisiología , Canales Iónicos/fisiología , Mecanotransducción Celular/fisiología , Tortugas/fisiología , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Diltiazem/química , Diltiazem/farmacología , Relación Dosis-Respuesta a Droga , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/química , Mecanotransducción Celular/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiologíaRESUMEN
Pathogenic bacteria utilise a number of mechanisms to cause disease in human hosts. Bacterial pathogens express a wide range of molecules that bind host cell targets to facilitate a variety of different host responses. The molecular strategies used by bacteria to interact with the host can be unique to specific pathogens or conserved across several different species. A key to fighting bacterial disease is the identification and characterisation of all these different strategies. The availability of complete genome sequences for several bacterial pathogens coupled with bioinformatics will lead to significant advances toward this goal.
Asunto(s)
Bacterias/patogenicidad , Adhesinas Bacterianas/fisiología , Bacterias/genética , Bacterias/inmunología , Adhesión Bacteriana/fisiología , Cápsulas Bacterianas/fisiología , Infecciones Bacterianas/etiología , Toxinas Bacterianas/química , Toxinas Bacterianas/clasificación , Pared Celular , Farmacorresistencia Bacteriana/fisiología , Humanos , Lipopolisacáridos/inmunología , Factor sigma/fisiología , Virulencia/fisiologíaRESUMEN
The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity of Salmonella enterica serovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed that Salmonella established infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection with Salmonella, 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore, Salmonella exhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection with Salmonella revealed significant differences in expression of interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor beta1 mRNA and prostaglandin E2 than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction.