In vitro secretion and activity profiles of matrix metalloproteinases, MMP-9 and MMP-2, in human term extra-placental membranes after exposure to Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Premature rupture of fetal membranes (PROM) complicated with intrauterine infection has been associated to alterations of the extracellular matrix (ECM) homeostasis. The aim of this work was to evaluate the integral/functional response of the amnion (AMN) and choriodecidua (CHD) to synthesis, secretion, and activity of MMP-2 and MMP-9 and of their inhibitors TIMP-1, -2, and -4, after stimulation with <it>Escherichia coli</it>.</p> <p>Methods</p> <p>Full-thickness membranes were mounted on a Transwell device, constituting two independent chambers, <it>Escherichia coli </it>(1×10 (6) CFU/mL) were added to either the amniotic or the choriodecidual face or to both. Secretion profiles of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-4 were quantified by ELISA and gelatinolytic activity by zymography. Immunoreactivity for MMP-2 and MMP-9 was revealed by immunohistochemistry and the collagen content was assessed by the hydroxyproline assay.</p> <p>Results</p> <p>Levels of MMP-9 in CHD and AMN increased 4- and 8-fold, respectively, after simultaneous infection. MMP-2 secreted to the medium by CHD increased a mean of 3 times after direct stimulation. Secretion profiles of TIMP-1, TIMP-2, and TIMP-4 remained without significant changes. Collagen content was significantly decreased (4-fold) in infected membranes, and was associated with loss of structural continuity and co-localization with immunoreactive forms of MMP-2 and MMP-9.</p> <p>Conclusions</p> <p>Infection of chorioamniotic membranes with <it>E. coli </it>induces an increase in the secretion of inactive forms and an association to ECM of active forms of MMP-2 and MMP-9 without changes in TIMP-1, -2, and -4. These changes could explain the significant decrease of collagen content and loss of structural continuity.</p

Light and focused ion beam microscopy workflow for resin-embedded tissues

Although the automated image acquisition with the focused ion beam scanning electron microscope (FIB-SEM) provides volume reconstructions, volume analysis of large samples remains challenging. Here, we present a workflow that combines a modified sample protocol of the classical transmission electron microscope with FIB-SEM volume imaging. The proposed workflow enables efficient 3D structural surveys of rabbit ovaries collected at consecutive developmental stages. The precise trimming of the region of interest adds the time dimension to the volume, constructing a virtual 4D electron microscopy. We found filopodia-like processes emitted by oocyte cysts allowing contact between oocytes not previously observed

In vitro secretion profiles of interleukin (IL)-1beta, IL-6, IL-8, IL-10, and TNF alpha after selective infection with Escherichia coli in human fetal membranes

<p>Abstract</p> <p>Background</p> <p>Chorioamniotic membranes infection is a pathologic condition in which an abnormal secretion of proinflammatory cytokines halts fetal immune tolerance. The aim of the present study was to evaluate the functional response of human chorioamniotic membranes, as well as the individual contribution of the amnion and choriodecidua after stimulation with Escherichia coli, a pathogen associated with preterm labor.</p> <p>Methods</p> <p>Explants of chorioamniotic membranes from 10 women (37–40 weeks of gestation) were mounted and cultured in a Transwell system, which allowed us to test the amnion and choriodecidua compartments independently. Escherichia coli (1 × 10 6 CFU/mL) was added to either the amniotic or the choriodecidual regions or both; after a 24-h incubation, the secretion of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10 in both compartments was measured using a specific ELISA. Data were analyzed by Kruskal-Wallis one-way analysis of variance.</p> <p>Results</p> <p>After stimulation with Escherichia coli, the choriodecidua compartment showed an increase in the secretion of IL-1beta (21-fold), IL-6 (2-fold), IL-8 (6-fold), and IL-10 (37-fold), regardless of which side of the membrane was stimulated; TNFalpha secretion augmented (22-fold) also but only when the stimulus was applied simultaneously to both sides. When the amnion was stimulated directly, the level of IL-1beta (13-fold) rose significantly; however, the increase in IL-8 secretion was larger (20-fold), regardless of the primary site of infection. TNFalpha secretion in the amnion compartment rose markedly only when Escherichia coli was simultaneously applied to both sides.</p> <p>Conclusion</p> <p>Selective stimulation of fetal membranes with Escherichia coli results in a differential production of IL-1beta, IL-6, TNFalpha, IL-8, and IL-10. These tissues were less responsive when the amnion side was stimulated. This is in agreement with the hypothesis that the choriodecidua may play a primary role during an ascending intrauterine infection, being the main barrier to progression of the infection into the amniotic cavity. Therefore, the tissue-specific capacities for the secretion of these immune modulators could be a determining factor for the degree of severity of the inflammation process in fetal membranes.</p

Nuevo avance hacia la clonación terapéutica humana: se reaviva la polémica

EstanciasLa clonación terapéutica como una opción para el tratamiento de pacientes con padecimientos genéticos recibió un nuevo impulso. Un equipo de la Universidad Nacional de Seúl, Corea del Sur, generó por primera vez una línea de células troncales derivadas de la masa celular interna de un blastocisto humano. El blastocisto fue desarrollado in vitro a partir de un cigoto “reconstruido” por la fusión de una célula somática del ovario con un ovocito enucleado. De manera que las células del blastocisto desarrollado poseen copias del genoma de la célula somática. Como las células troncales obtenidas son genéticamente idénticas a la célula somática, se asume que pudieran ser usadas para transplantarse ya que el rechazo inmunológico sería nulo o muy leve. Si además de células ováricas usadas por el equipo coreano, se derivan células troncales clonadas a partir de otras células de individuos adultos, el camino hacia una clonación terapéutica confiable estará abierto. Sin embargo, el manejo de ovocitos humanos “reconstruídos” para generar blastocistos plantea cuestiones éticas porque de ser implantados en el útero de una mujer, podrían desarrollarse como individuos clonados. Sin embargo, hay una importante diferencia entre las clonaciones terapéutica y reproductiva en términos de “intencionalidad”. Mientras la primera intenta clonar células para aliviar a un paciente, en la segunda se busca clonar a un individuo. Como en ambos tipos de clonación se requiere del ovocito humano, la discusión puede centrarse en la potencialidad única de esa célula para desarrollar a un ser humano. En el presente artículo expongo una opinión al respecto de acuerdo con el actual contexto biológicoTherapeutic cloning as an alternative clinical method to treat genetic illness has received a new impulse. In South Corea, a team working in the National University of Seul has established for the first time a line of stem cells derived from the inner cell mass of a cloned human blastocyst. The blastocyst developed from a “reconstructed” egg made by fusing an enucleated oocyte with a somatic ovarian cell. Thus, the cells of the developed blastocyst contained copies of the somatic cell genome. Since the derived stem cells are genetically identical to the somatic cell, it is assumed that they can be used for transplantation because the immunological rejection may be null or highly reduced. If besides ovarian somatic cells used by the Corean team, stem cells can also be derived from other adult somatic cells, the way for a reliable therapeutic cloning in humans will be open. However, handling human oocytes “reconstructed” posse ethical concerns since they may develop as a cloned baby if transferred to a woman womb. Thus, distinction between therapeutic and reproductive cloning is in terms of “intentionality”. While the former intends to clone cells of a patient, the latter seeks to clone individuals. Since for both, therapeutic and reproductive cloning human oocytes are required, discussion is centered on the potential of these cells to develop as human beings. In this article a personal opinion is given considering the actual biological contex

Bases moleculares de la determinación sexual en mamíferos

Se correlacionan conceptos clásicos de la diferenciación sexual con mecanismos moleculares de la determinación sexual en mamíferos. El paradigma de Jost estableció que la diferenciación sexual fetal depende de la actividad endócrina de los testículos. En la gónada embrionaria indiferenciada se establecen redes moleculares a partir de vías alternativas de expresión que determinan la formación de ovarios o testículos. Tomando al ratón como modelo, describimos el hallazgo de varios genes que se activan o reprimen a partir de la expresión del Sry en los machos y las vías alternas encontradas en las hembras. Concluimos que todavía queda por conocerse el alcance que tienen los datos del ratón como modelo para extrapolarse a otros mamíferos incluido el humano

Consideraciones generales en el establecimiento del sexo en mamíferos

Establishment of sex in mammals occurs at the moment of fertilization, however, the determination and sexual differentiation of the gonad is required in order to establish the sexual phenotype of the individual. Since the discovery of the Sry gene as the testis-determining factor, progress has been made in understanding the mechanisms that lead to testicular development and establishment of the male phenotype. Although the female phenotype is acquired even in the absence of the gonad, differentiation and ovarian maturation are required for the establishment of secondary sexual characteristics. This work aims to describe the mechanisms involved in sexual determination of the gonad and integrate them with the histological aspects that contribute to gonadal sex differentiation. Once the gonadal sex is established, molecular aspects involved in the masculinization of the individual are addressed. Finally, a brief review of the pathological processes resulting from the alteration of the establishment of sex in humans is undertaken. El establecimiento del sexo en mamíferos ocurre al momento de la fertilización, sin embargo, se requiere de la determinación y diferenciación sexual de la gónada para que se establezca el fenotipo sexual del individuo. A partir del descubrimiento del gen Sry como el factor determinante del testículo se ha avanzado en conocer los mecanismos que conducen al desarrollo testicular y al establecimiento del fenotipo masculino. Y aunque el fenotipo femenino se adquiere aun en ausencia de la gónada, se requiere de la diferenciación y maduración ovárica para la adquisición de los caracteres sexuales secundarios. Este trabajo tiene como objetivo describir  los mecanismos involucrados en la determinación sexual de la gónada e integrarlos con los aspectos histológicos que contribuyen con la diferenciación sexual gonadal. Una vez establecido el sexo gonadal se abordan aspectos moleculares involucrados en la masculinización del individuo. Finalmente se hace una breve revisión sobre los procesos patológicos resultantes de la alteración del establecimiento del sexo en humanos.