Listeria monocytogenes induces mast cell extracellular traps.

Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when ß-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.

Endothelial colony-forming cells: Biological and functional abnormalities in patients with recurrent, unprovoked venous thromboembolic disease.

INTRODUCTION: Endothelial cells (ECs) are an important component of the blood coagulation system because it maintains blood fluid. Because in patients with venous thromboembolic disease (VTD) a thrombophilic condition is not found sometimes, we investigated if endothelial colony-forming cells (ECFCs) from these patients have biological and functional abnormalities. PATIENTS AND METHODS: Human mononuclear cells (MNCs) were obtained from peripheral blood from patients with VTD and controls to obtain ECFCs. These cells were assayed for their immunophenotype and electron microscopy characteristics and their ability to form capillary-like structures and to produce pro-inflammatory and pro-angiogenic cytokines and reactive oxygen species (ROS). RESULTS: ECFCs appeared at 7 and 21 days of culture in VTD patients and controls, respectively. ECFCs increased 8-fold in patients and emerged 1 week earlier. No differences in the size of the colonies of ECFCs were found. Numbers and time of appearance of ECFCs was different between groups. ECFC-derived ECs (ECFC-ECs) of both groups expressed CD31, CD34, CD146, and CD-309 but none expressed CD45, CD14, or CD90. Interest CD34 was highly expressed in ECFC-ECs from patients. In both groups, ECFC-ECs showed similar capacity to form capillary-like structures but ECFC-ECs from patients had significant abnormalities in the mitochondrial membrane. We found a significant increase in ROS production in ECFC-ECs from patients. There were significant differences in cytokine profiles between VTD patients and controls. CONCLUSIONS: We found a dysfunctional state in ECFC from VTD patients resembling some characteristics of dysfunctional ECs. These findings may help to understand some pathophysiological aspects of VTD.

Prolonged exposure to neutrophil extracellular traps can induce mitochondrial damage in macrophages and dendritic cells.

Neutrophils are one the earliest, crucial innate defenses against innumerable pathogens. Their main microbicidal activities include phagocytosis and degranulation, with many pharmacologically active molecules contributing to inflammation. Recently, a novel antimicrobial mechanism was discovered; the Neutrophil Extracelullar Traps (NETs) formed by extrusion of DNA and associated molecules (histones, elastase, antimicrobial peptides, among others) which trap and kill microorganisms. Since NETs were recently described, research has focused on their induction and microbicidal properties, and recently on disease involvement. However, the functional consequences of NETs interacting with other immune cells, either resident or recruited during early inflammation, have not been assessed. We therefore investigated the consequences of exposing two major APCs, macrophages (Mfs) and conventional Dendritic Cells (cDCs) to NETs. Our data revealed that at early times (30 min), both Antigen Presenting Cells (APCs) showed induction of important costimulatory molecules (CD80, CD86). Unexpectedly, however, at later times (6 and 24 hours) NETs apparently triggered a cell death process in these APCs by a caspase- and Apoptosis induced factor (AIF)-dependent pathway, suggesting mitochondrial damage. By rhodamine-123 labelling we found that in both APCs, relatively prolonged exposure to NETs or their components importantly decreased the mitochondrial membrane potential. Ultrastructural analysis confirmed mitochondrial alterations in both APCs. Our results would suggest that early in inflammation, NETs can activate the two main APCs (Mfs and cDCs), but as the process continues, NETs can then initiate apoptosis of these cells through mitochondrial harm. Conceivable, this "late" induction of cell death in these two APCs might start limiting an ongoing inflammatory process to control it.

Interaction between pathogenic bacteria and intrauterine leukocytes triggers alternative molecular signaling cascades leading to labor in women.

Increased risk of preterm labor has been linked to cervicovaginal infection with Ureaplasma urealyticum and group B streptococci. Although various experimental models have been developed to study the role of amniochorion infection in preterm labor, they typically exclude the initial interaction between intrauterine leukocytes (recruited from decidual vessels into the avascular fetal membranes) and infecting bacteria. In this work, we ascertained whether inflammatory molecules secreted by bacterium-activated intrauterine leukocytes stimulate the amniochorion production of mediators involved in human labor. Using a two-step process beginning with placental circulating leukocytes as a proxy for intrauterine leukocytes, we found that coincubation of amniochorion explants with plasma from placental whole blood preincubated with group B streptococci resulted in a significant increase in tumor necrosis factor alpha (TNF-α) and matrix metalloproteinase 9 (MMP-9) levels in tissue. Extensive changes in the connective tissue arrangement and a decrease in collagen content demonstrated the degradation of the extracellular matrix following this treatment. In contrast, plasma from blood preconditioned with U. urealyticum induced a highly significant secretion of interleukin-1ß (IL-1ß) and prostaglandin E(2) (PGE(2)) by the amniochorion without changes in the extracellular matrix organization or content. These data demonstrate that group B streptococci induce degradation of the amniochorion as a result of MMP-9 production, probably via TNF-α, whereas U. urealyticum stimulates the secretion of PGE(2), probably via IL-1ß, potentially stimulating myometrial contraction. Our study provides novel evidence that the immunological cells circulating within the uterine microenvironment respond differentially to an infectious agent, triggering alternative molecular signaling pathways leading to human labor.

Initial characterization of the microenvironment that regulates connective tissue degradation in amniochorion during normal human labor.

Extracellular matrix degradation in fetal membranes leading to its rupture is coupled to myometrial activity and cervical ripening during human normal labor. Mechanisms which modulate collagen degradation in amniochorion during labor have not been elucidated. Initial characterization of the effect of different blood compartments on connective tissue degradation in amniochorion during human labor was explored. Amniochorion explants were stimulated with plasma of maternal venous blood, umbilical cord blood or placental blood, obtained from women with pregnancies at term, with or without labor. MMP-2 and MMP-9 activities were quantified in conditioned media by gelatin-zymography as an index of connective tissue degradation. Collagen content was measured in tissue explants and collagen fibrils distribution was examined by electron microscopy. Placental plasma from term pregnancies, with or without labor, is enriched with soluble signals that enhance the in vitro MMP-9 production by amniochorion. Accompanying ultrastructural distortion of collagen fibers and demonstration of collagen degradation fragments confirmed induction of extracellular matrix degradation. Control experiments in which MMP-9 activity was blocked with TIMP-1 resulted in inhibition of all the above mentioned changes. These results suggest that placental intervillous space is a functional compartment in which mediators capable to induce collagen degradation in amniochorion are selectively expressed during human labor.

Histological study of the proximal and distal segments of the embryonic outflow tract and great arteries.

The normal development of the ventricular outlets and proximal region of the great arteries is a controversial subject. It is known that the conus, truncus arteriosus (truncus), and aortic sac participate; however, there are some doubts as to the actual prospective fate of the truncus. Some authors propose that it gives origin to the proximal region of the great arteries and that the myocardial cells of its wall become smooth muscle. Nevertheless, others think that the truncus only forms the arterial valve apparatus and that therefore the myocardial cells transform into fibroblasts. As a first approach to beginning to elucidate which process occurs, the aim of this article was to study the histological changes in the wall of these components of the developing heart in chick embryos whose hearts had been labeled at the truncoconal boundary at stage 22HH, tracing the changes up to stage 36HH. Also, the histological constitution of the wall of the pulmonary arterial trunk and its valve apparatus were studied in the posthatching and adult hearts of chickens and rats. The conus and truncus walls were always encircled by a myocardial sleeve from the outset of their development. Between stages 26HH to 28HH, the truncal myocardial cells adjacent to the mesenchymal tissue of the ridges began to lose cell-to-cell contacts and invaded the extracellular matrix. At stage 24HH, the aortic sac began to project into the pericardial cavity and became divided into two channels by the aortic-pulmonary septum at stage 26HH. The wall of the aortic sac is mostly constituted by a compact mesenchymal tissue. Initially, it does not have smooth muscle but this starts to appear at stage 30HH. The insertion ring of the valves, a broad structure, was formed by mesenchymal tissue. Both structures were always covered by a myocardial sleeve. The leaflets developed from the truncal ridges, the segment immediately proximal to the aortic sac. Our results indicate that the proximal region of the pulmonary and aortic arteries do not originate from the truncus arteriosus; rather, we found that they take origin from the aortic sac. Thus, our findings agree with the proposal that the myocardial cells of the external sleeve of the truncus become fibroblastic and suggest that the insertion ring of the arterial valves has a dual origin: fibroblasts produced by truncal myocardial transdiferentiation and the mesenchymal tissue of the proximal region of the truncal ridges, while the leaflets have their origin from the truncal ridges. We discuss the fact that, because the truncus arteriosus does not give origin to the trunks of the aortic and pulmonary arteries, it may be necessary to modify terminology. Based on our results, together with the new findings obtained by in vivo labeling, immunostaining, a chimeric approach, and ultrastructural studies, we propose a developmental model that correlates the fate of the conus, truncus, and aortic sac with the normal morphogenesis of the ventricular outlet tracts and the trunks of the great arteries. (c) 2005 Wiley-Liss, Inc.

[Changes in the systemic immunologic response in association with endometriosis using an animal model]. / Alteración de la respuesta inmunológica sistémica en asociación con endometriosis, empleando un modelo de estudio animal.

INTRODUCTION: Endometriosis constitutes the growth of endometrial tissue in a place other than the uterine cavity. Its etiopathogenesis is unknown, although there is some evidence associating it with the decrease of cytotoxic activity in the immunological system. OBJECTIVE: Evaluating the relationship between the development of ectopic endometrial tissue and the immunological status, and enumerating lymphocyte subpopulations and cytokine synthesis in T lymphocytes, using a murine endometriosis model. METHODOLOGY: Spleen lymphocytes isolated from two study groups of 10 female mice of the Balb/c strain that had been submitted to the surgical implantation of autologous endometrial tissue in the peritoneal cavity, and sacrificed after 5 (group I) and 8 (group II) weeks, were incubated--or not--with PMA/lonomicine. Lymphocyte T numbers and their cytokine production were determined by flow cytometry. RESULTS: A lower dispersion of the ectopic tissue growth value was observed in group II (24% vs. 42%). A smaller population of cytotoxic T lymphocytes and a greater IL-4 production in the stimulated cells of the study group (p < 0.05) were observed, as compared to the control group. CONCLUSIONS: The presence of endometrial tissue in the uterine cavity decreases the amount of cytotoxic T lymphocytes and increases IL-4 production in total T lymphocytes, suggesting a modulation of the systemic immunological response to TH-2.

Association of type II apoptosis and 92-kDa type IV collagenase expression in human amniochorion in prematurely ruptured membranes with tumor necrosis factor receptor-1 expression.

OBJECTIVE: We assessed the presence of tumor necrosis factor receptor-1 (TNF-R1), apoptosis, and simultaneous expression of 92-kDa collagenase type IV (MMP-9) in samples of human chorioamnion from women with premature rupture of membranes (PROM). METHODS: Amniotic membranes from women who underwent normal labor, cesarean delivery, or had PROM at term were studied by immunohistochemistry for localization of TNF-R1 and R2. Transmission electron microscopy and DNA fragmentation analyses by agarose gel electrophoresis were performed to identify apoptosis characteristics. Zymography and in situ zymography were used to assess gelatinolytic activity. RESULTS: We found that TNF-R1 was abundant in membranes from subjects who had normal labor and very abundant in those who had PROM. By contrast TNF-R2 was abundant only in membranes from subjects who had cesarean delivery. Gelatinolytic activity was associated with extracellular matrix rather than cells and was higher in extracts from fetal membranes from PROM and normal labor than in extracts obtained from cesarean deliveries. Transmission electron microscopy of fetal membranes from PROM revealed ultrastructural characteristics in amnion epithelium consistent with type II apoptosis. DNA laddering in agarose gel electrophoresis corroborated results from DNA fragmentation. CONCLUSION: During PROM the fetal membranes undergo type II apoptosis and extracellular matrix degradation in association with TNF-R1 expression.

Estudio de la osteoporosis y osteopenia por medio de radiología, densitometría, morfometría y microscopia de barrido en la tercera vértebra lumbar en una muestra de hombres y mujeres en la ciudad de México(AU) / Radiologic, densitometry, morphometric and sacanning electron microscopy study of osteoporosis or osteopenia ot the third lumbar vertebra in to female and male of the Mexico city

Objetivo. Determinar en la tercera vértebra lumbar de un grupo de hombres y mujeres de la Ciudad de México la densidad ósea estableciendo y correlacionando las características morfométricas del cuerpo vertebral, por medio de técnicas específicas y por detectar grupos con alteraciones de osteoporosis y osteopenia. Metodología. El estudio se realizó en la tercera vértebra lumbar de 23 cadáveres del Servicio Médico Forense, 8 mujeres de 31 a 72 años y 15 hombres de 25 a 62 años. Los estudios efectuados fueron: radiológicos, densitometría ósea y análisis de imagen, en la que se determinó espesor de las trabéculas verticales y horizontales. Con microscopia electrónica de barrido por medio de la Técnica de rayos X se determinó en forma semicuantitativa la presencia de calcio, fósforo, magnesio y sodio y en forma cualitativa la distribución de calcio en una área determinada del cuerpo vertebral mediante la técnica de energía dispersada de rayos X. Resultados. Estudio radiológico en el grupo de mujeres ninguna vértebra estudiada fue normal, todas ellas presentaron cambios degenerativos, de éstas cinco con trabeculación aumentada y tres con evidencia de fractura, con respecto al grupo de hombres siete fueron normales y ocho casos con cambios degenerativos de éstos seis presentaron densidad ósea con densitometría con DEXA en el grupo de mujeres sólo una se determinó normal, tres osteopenia y cinco con osteoporosis, en el grupo de varones cinco fueron normales, cinco con osteopenia y cinco con osteoporosis. En la determinación del mapeo de calcio por medio del microscopio de barrido, se pudo observar la distribución de calcio, en los casos normales se observó su distribución se presenta menos densa existiendo áreas en las que el calcio se encuentra disminuido o bien se encuentra ausente. En el estudio morfométrico se determinó el espesor trabecular de nueve vértebras, de cinco mujreses y cuatro hombres, en el grupo de mujeres, una presentó un espesor trabecular promedio normal de 222.1µm, en las cuatro restantes con osteoporosis el rango fue de 126.3 a 156.2 µm, en el grupò de varones se encontraron dos normales con un espesor trabecular promedio de 249.7 µm...

Vaginosis bacteriana. Relación de la flora vaginal con las células epiteliales de vagina, con diferente tratamiento. Estudio ultraestructural / Bacterial vaginoss. relation of vaginal flora with epithelial cells of vagina, with different treatment. Ultraestrucural study

Los estudios relativos al tratamiento contra la vaginosis bacteriana han sido sobre efectividad probando concentraciones y tiempos del tratamiento. En el presente estudio analizamos la relación morfológica de la flora vaginal con el epitelio de la vagina, en mujeres con vaginosis bacteriana, en pre-tratamiento, primer postratamiento empleando metronidazol oral y fosfato de clindamicina intravaginal. De las 20 pacientes con vaginosis bacteriana, a 10 se les prescribió metronidazol oral 500 mg/2 al día X7 días; a las restantes, clindamicina 2 por ciento intravaginal una aplicación diaria por siete días. A las parejas se les suministró metronidazol en la dosis señalada. Las muestras obtenidas se procesaron mediante técnicas habituales para microscopia electrónica de transmisión. Se realizaron cortes ultrafinos de 100 mm de grosor y cortes seriados del mismo grosor. En pre-tratamiento las prolongaciones celulares ocasionaron mayor adhesión de las bacterias; asimismo, se encontraron restos de uniones celulares con bacterias adheridas. Se encontró penetración de algunas bacterias a las células epiteliales, corroborándose con cortes seriados y destacando la superposición de éstas en las células. Esta penetración se encontró en cinco casos y persistió en el primero y segundo postratamientos. En el primer postratamiento, se encontraron grupos celulares sin bacteria, la presencia de lactobacilos fue baja y aumentó posteriormente. La presencia de levaduras se presentó en el primer postratamiento y en algunos casos persistió. Es importante diferenciar la(s) bacteria(s) intracelulares e inferir las características relacionadas con la penetración bacteriana, para una adecuada prescripción y un mejor aprovechamiento de los fármacos previniendo posibles daños. La presencia de bacterias intracelularmente puede ser una de las causas de reincidencia de vaginosis bacteriana

Análisis ultraestructural de placentas con villitis. Estudio retrospectivo / Ultrastructural analysis of villitis placentae. a retrospective study

La villitis es una inflamación crónica focal o multifocal de las vellocidades coriónicas con infiltración de células mononucleares y áreas de necrosis fibrinoide la cual puede ser producida por infección viral (VIV) o bien puede ser de etiología desconocida (VED). En el presente estudio se realizó un examen morfológico a nivel ultraestructural de 11 placentas con diagnóstico de villitis con el objetivo de establecer una comparación de tipo descriptivo entre las VIV y las VED. Las biopsias fueron procesadas con las técnicas convencionales para microscopía óptica y electrónica. Las observaciones ultraestructurales permitieron confirmar la presencia de partículas virales en cuatro casos, mientras que en los restantes siete no se detectó ningún tipo de virus. Se notó ausencia o disminución focal en las microvellosidades lo cual estuvo asociado con la presencia de necrosis fibrinoide en el estroma y clínicamente a cuatro partos pretérmino y un óbito. En ambos tipos de villitis se encontraron alteraciones en el trofoblasto como engrosamiento de la membrana basal asociado con depósito de material electrodenso similar a calcio; también se observó vacuolización y edema en el estroma de las vellosidades. En algunos casos se notó la presencia de depósitos focales de fibrina asociado con zonas de necrosis en el estroma, precipitados de calcio y cuerpos mielínicos en el sinciotrofoblasto. En los casos de infección viral (dos por CMV y dos positivos para TORCH) los vasos fetales mostraron obliteración y/o trombos. En estos casos se detectaron partículas virales con incremento de las células de Hofbauer y calcificaciones en la membrana basal. En conclusión podemos decir que en ambos tipos de villitis existe una reacción inmunológica que produce la pérdida o disminución de las microvellosidades del trofoblasto con infiltración de células mononucleares y edema. Estas alteraciones reducen importantemente el intercambio materno-fetal de gases, nutrientes, hormonas etc., lo cual puede ser la causa del retraso en el crecimiento, la inmadurez o la muerte del feto

Infección por Gardnerella vaginalis en parejas heterosexuales: estudio ultraestructural en células de descamación del epitelio estratificado / Gardnerella vaginalis infection in heterosexual couples: ultrastructural al study in descamation cells of stratified epithelium

La vaginosis bacteriana es una de las infecciones más frecuentes en la edad reproductiva de la mujer. La flora normal de lactobacilos es sustituída por concentraciones relativamente elevadas de Gardnerella vaginalis (GV), bacteroides anaerobios, Mobiluncus y Mycoplasma. El objetivo de este trabajo es hacer un análisis morfológico de los posibles mecanismos de adhesión y penetración de GV en la pareja heterosexual tanto en el epitelio escamoso de la pared vaginal como en el líquido seminal. Para cumplir con nuestro objetivo se estudiaron 10 parejas con cultivo de GV que tenían de 3 a 4 días de abstenencia sexual. Las mujeres presentaron al menos 3 de los 4 criterios de Amsel. Se obtuvieron muestras de las paredes laterales de la vagina y de líquido seminal, éstas se dividieron en dos partes: 1) para realizar cultivos para GV, Ureaplasma urealyticum y Mycoplasma hominis, y 2)para un análisis ultraestructural. Las muestras fueron procesadas con las técnicas habituales para microscopía electrónica. En las células vaginales se encontraron bacterias semejantes a GV en forma libre, adherencias a la membrana plasmática y dentro del citoplasma celular. En el líquido seminal se encontraron numerosas células ureterales de descamación que presentaron, al igual que en la mujer, bacterias en forma libre, adherida a la membrana plasmática y dentro del citoplasma. En 4 casos se encontraron bacterias semejantes a mycoplasma y un caso con partículas que sugieren citomegalovirus. En este trabajo se concluye que: 1) el varón presenta células uretrales semejantes a células guía" de la vagina de la mujer 2) la GV coloniza el epitelio ureteral del varón, 3) el varón es capaz de infectar y/o reinfectar a la mujer