RESUMEN
In this study, we have investigated innate immune activation capacity and metabolic features of a population of P. aeruginosa PAO1 phage-resistant mutants with diverse genetic modification (large genomic deletions and point mutations) arising after exposure to phages targetting lipopolysaccharide (LPS) or Type-4 pili (T4P). Deletions led to the loss of genes involved in LPS synthesis, cell envelope permeability, efflux systems, biofilm production, oxidative stress tolerance, and DNA repair. Loss of LPS O antigen resulted in bacterial sensitivity to serum complement and stimulation of inflammatory cascades but did not cause increased phagocytosis, while T4P phage-resistant mutants were more effectively phagocytized than LPS-defective mutants. Changes in the utilization of different carbon, nitrogen, sulphur, and phosphorus sources were identified, especially in mutants where the two phage DNA persisted in the bacterial population (pseudolysogeny). However, the metabolic changes did not directly correlate with single-gene mutations or the large gene deletions, suggesting they reflect adaptive changes to the gene modifications that arise during the selection of resistant mutants. In contrast, phage-resistant mutants were susceptible to humoral innate immune responses, suggesting that phage resistance may be a beneficial outcome of phage therapy.
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Bacteriófagos , Pseudomonas aeruginosa/metabolismo , Lipopolisacáridos , Bacterias/metabolismo , Inmunidad Innata , MetabolomaRESUMEN
Macrophages are vital inhabitants of the developing heart. Nonetheless, their key role is not limited to prenatal processes, as embryo-derived macrophages govern the pool of cardiac macrophages also postnatally. Namely, embryonic cardiac macrophages are of yolk sac-, embryonic monocyte-, and heart-tissue origin. They persist, self-renew and/or are gradually replaced by blood monocytes and assume microenvironment-dependent macrophage phenotypes both in the pre- and postnatal heart. Still, it is during embryonic development that cardiac macrophages gain tissue-specific phenotypes and multifunctional diverse properties. Currently, with the emergence of newer research methods, novel facts about embryonic macrophage ontogeny, lifecycle, and repertoire of functions have been revealed. Meeting the high interest in cardiac macrophages, we present this up-to-date overview of embryonic cardiac macrophages, emphasizing the fundamental concepts and discrepancies related to macrophage characteristics, current research gaps, and potential future developments in this field.
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Macrófagos , Monocitos , Embrión de Mamíferos , Corazón , Saco VitelinoRESUMEN
Bacterial surface structures of a proteinic nature and glycoconjugates contribute to biofilm formation and provide shields to host defense mechanisms (e.g., the complement system and phagocytosis). A loss or alteration of these molecules, leading to phage resistance, could result in fewer virulent bacteria. In this study, we evaluate the biology and phenotype changes in Pseudomonas aeruginosa PAO1 phage-resistant clones, which emerge in phage-treated biofilms. We characterize these clones for phage-typing patterns, antibiotic resistance, biofilm formation, pathogenicity, and interactions with the innate immune system. Another important question that we address is whether phage-resistant mutants are also generated incidentally, despite the phage treatment-selective pressure, as the natural adaptation of the living biofilm population. It is found that the application of different phages targeting a particular receptor selects similar phage resistance patterns. Nevertheless, this results in a dramatic increase in the population heterogeneity, giving over a dozen phage-typing patterns, compared to one of the untreated PAO1 sessile forms. We also confirm the hypothesis that "phage-resistant bacteria are more susceptible to antibiotics and host-clearance mechanisms by the immune system". These findings support phage application in therapy, although the overall statement that phage treatment selects the less virulent bacterial population should be further verified using a bigger collection of clinical strains.
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Farmacorresistencia Microbiana/genética , Fagos Pseudomonas/genética , Pseudomonas aeruginosa/virología , Antibacterianos/farmacología , Bacteriófagos/genética , Biopelículas/crecimiento & desarrollo , Farmacorresistencia Microbiana/fisiología , Humanos , Terapia de Fagos/métodos , Fagocitosis/genética , Fenotipo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , VirulenciaRESUMEN
The role of cardiac tissue macrophages (cTMs) during pre- and postnatal developmental stages remains in many aspects unknown. We aimed to characterize cTM populations and their potential functions based on surface markers. Our in situ studies of immunostained cardiac tissue specimens of murine fetuses (from E11to E17) revealed that a significant number of embryonic cTMs (phenotyped by CD45, CD68, CD64, F4/80, CD11b, CD206, Lyve-1) resided mostly in the subepicardial space, not in the entire myocardial wall, as observed in adult individuals. cTMs accompanied newly developed blood and lymphatic vessels adhering to vessel walls by cellular processes. A subpopulation of CD68-positive cells was found to form accumulations in areas of massive apoptosis during the outflow tract remodeling and shortening. Flow cytometry analysis at E14 and E17 stages revealed newly defined three subpopulations:CD64low, CD64highCD206-and CD64highCD206+. The levels of mRNA expression for genes related to regulation of angiogenesis (VEGFa, VEGFb, VEGFc, bFGF), lymphangiogenesis (VEGFc) and extracellular matrix (ECM) remodeling (MMP13, Arg1, Ym1/Chil3, Retlna/FIZZ1) differed among the selected populations and/or embryonic stages. Our results demonstrate a diversity of embryonic cTMs and their tissue-specific locations, suggesting their various potential roles in regulating angiogenesis, lymphangiogenesis and ECM remodeling.
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Matriz Extracelular/metabolismo , Linfangiogénesis , Macrófagos/metabolismo , Modelos Biológicos , Miocardio/metabolismo , Neovascularización Fisiológica , Animales , Desarrollo Fetal , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Miocardio/citologíaRESUMEN
Bacterial biofilm prevention and eradication are common treatment problems, hence there is a need for advanced and precise experimental methods for its monitoring. Bacterial resistance to antibiotics has resulted in an interest in using a natural bacterial enemy-bacteriophages. In this study, we present the application of quartz tuning forks (QTF) as impedance sensors to determine in real-time the direct changes in Pseudomonas aeruginosa PAO1 biofilm growth dynamics during Pseudomonas phage LUZ 19 treatment at different multiplicities of infection (MOI). The impedance of the electric equivalent circuit (EEC) allowed us to measure the series resistance (Rs) corresponding to the growth-medium resistance (planktonic culture changes) and the conductance (G) corresponding to the level of QTF sensor surface coverage by bacterial cells and the extracellular polymer structure (EPS) matrix. It was shown that phage impacts on sessile cells (G dynamics) was very similar in the 10-day biofilm development regardless of applied MOI (0.1, 1 or 10). The application of phages at an early stage (at the sixth h) and on three-day biofilm caused a significant slowdown in biofilm dynamics, whereas the two-day biofilm turned out to be insensitive to phage infection. We observed an inhibitory effect of phage infection on the planktonic culture (Rs dynamics) regardless of the MOI applied and the time point of infection. Moreover, the Rs parameter made it possible to detect PAO1 population regrowth at the latest time points of incubation. The number of phage-insensitive forms reached the level of untreated culture at around the sixth day of infection. We conclude that the proposed impedance spectroscopy technique can be used to measure the physiological changes in the biofilm matrix composition, as well as the condition of planktonic cultures in order to evaluate the activity of anti-biofilm compounds.
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Biopelículas/crecimiento & desarrollo , Espectroscopía Dieléctrica/métodos , Fagos Pseudomonas/fisiología , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/virología , Medios de Cultivo , Matriz Extracelular de Sustancias Poliméricas/fisiologíaRESUMEN
The emergence of phage-resistant mutants is a key aspect of lytic phages-bacteria interaction and the main driver for the co-evolution between both organisms. Here, we analyze the impact of PA5oct jumbo phage treatment on planktonic/cell line associated and sessile P. aeruginosa population. Besides its broad-spectrum activity and efficient bacteria reduction in both airway surface liquid (ASL) model, and biofilm matrix degradation, PA5oct appears to persist in most of phage-resistant clones. Indeed, a high percentage of resistance (20/30 clones) to PA5oct is accompanied by the presence of phage DNA within bacterial culture. Moreover, the maintenance of this phage in the bacterial population correlates with reduced P. aeruginosa virulence, coupled with a sensitization to innate immune mechanisms, and a significantly reduced growth rate. We observed rather unusual consequences of PA5oct infection causing an increased inflammatory response of monocytes to P. aeruginosa. This phenomenon, combined with the loss or modification of the phage receptor, makes most of the phage-resistant clones significantly less pathogenic in in vivo model. These findings provide new insights into the general knowledge of giant phages biology and the impact of their application in phage therapy.
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Biopelículas/crecimiento & desarrollo , Plancton/microbiología , Fagos Pseudomonas/fisiología , Pseudomonas aeruginosa/virología , Mutación , Terapia de Fagos , Fenotipo , Fagos Pseudomonas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , VirulenciaRESUMEN
The virulence of bacterial outer membrane vesicles (OMVs) contributes to innate microbial defense. Limited data report their role in interspecies reactions. There are no data about the relevance of OMVs in bacterial-yeast communication. We hypothesized that model Moraxella catarrhalis OMVs may orchestrate the susceptibility of pathogenic bacteria and yeasts to cationic peptides (polymyxin B) and serum complement. Using growth kinetic curve and time-kill assay we found that OMVs protect Candida albicans against polymyxin B-dependent fungicidal action in combination with fluconazole. We showed that OMVs preserve the virulent filamentous phenotype of yeasts in the presence of both antifungal drugs. We demonstrated that bacteria including Haemophilus influenza, Acinetobacter baumannii, and Pseudomonas aeruginosa coincubated with OMVs are protected against membrane targeting agents. The high susceptibility of OMV-associated bacteria to polymyxin B excluded the direct way of protection, suggesting rather the fusion mechanisms. High-performance liquid chromatography-ultraviolet spectroscopy (HPLC-UV) and zeta-potential measurement revealed a high sequestration capacity (up to 95%) of OMVs against model cationic peptide accompanied by an increase in surface electrical charge. We presented the first experimental evidence that bacterial OMVs by sequestering of cationic peptides may protect pathogenic yeast against combined action of antifungal drugs. Our findings identify OMVs as important inter-kingdom players.
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Bacterias/patogenicidad , Membrana Celular/metabolismo , Proteínas del Sistema Complemento/farmacología , Vesículas Extracelulares/metabolismo , Péptidos/farmacología , Suero/metabolismo , Levaduras/patogenicidad , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Cationes , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular/efectos de los fármacos , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/ultraestructura , Fluconazol/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Moraxella/metabolismo , Polimixina B/farmacología , Electricidad Estática , Virulencia/efectos de los fármacos , Levaduras/efectos de los fármacos , Levaduras/crecimiento & desarrolloRESUMEN
The environment exerts strong influence on microbes. Adaptation of microbes to changing conditions is a dynamic process regulated by complex networks. Pseudomonas aeruginosa is a life-threating, versatile opportunistic and multi drug resistant pathogen that provides a model to investigate adaptation mechanisms to environmental changes. The ability of P. aeruginosa to form biofilms and to modify virulence in response to environmental changes is coordinated by various mechanisms including two-component systems (TCS), and secondary messengers involved in quorum sensing (QS) and c-di-GMP networks (diguanylate cyclase systems, DGC). In this review, we focus on the role of c-di-GMP during biofilm formation. We describe TCS and QS signal cascades regulated by c-di-GMP in response to changes in the external environment. We present a complex signaling network dynamically changing during the transition of P. aeruginosa from the free-living to sessile mode of growth.
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Biopelículas , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/fisiología , Transducción de Señal/fisiología , GMP Cíclico/metabolismo , Pseudomonas aeruginosa/crecimiento & desarrolloRESUMEN
The proepicardium (PE) is a transitory extracardiac embryonic structure which plays a crucial role in cardiac morphogenesis and delivers various cell lineages to the developing heart. The PE arises from the lateral plate mesoderm (LPM) and is present in all vertebrate species. During development, mesothelial cells of the PE reach the naked myocardium either as free-floating aggregates in the form of vesicles or via a tissue bridge; subsequently, they attach to the myocardium and, finally, form the third layer of a mature heart-the epicardium. After undergoing epithelial-to-mesenchymal transition (EMT) some of the epicardial cells migrate into the myocardial wall and differentiate into fibroblasts, smooth muscle cells, and possibly other cell types. Despite many recent findings, the molecular pathways that control not only proepicardial induction and differentiation but also epicardial formation and epicardial cell fate are poorly understood. Knowledge about these events is essential because molecular mechanisms that occur during embryonic development have been shown to be reactivated in pathological conditions, for example, after myocardial infarction, during hypertensive heart disease or other cardiovascular diseases. Therefore, in this review we intended to summarize the current knowledge about PE formation and structure, as well as proepicardial cell fate in animals commonly used as models for studies on heart development. Anat Rec, 302:893-903, 2019. © 2018 Wiley Periodicals, Inc.
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Transición Epitelial-Mesenquimal/fisiología , Mesodermo/embriología , Pericardio/embriología , Células Madre Pluripotentes/fisiología , Animales , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Células Epiteliales/fisiología , Fibroblastos/fisiología , Humanos , Mesodermo/citología , Miocitos del Músculo Liso/fisiología , Pericardio/citología , Especificidad de la EspecieRESUMEN
Pseudomonas phage LKA1 of the subfamily Autographivirinae encodes a tailspike protein (LKA1gp49) which binds and cleaves B-band LPS (O-specific antigen, OSA) of Pseudomonas aeruginosa PAO1. The crystal structure of LKA1gp49 catalytic domain consists of a beta-helix, an insertion domain and a C-terminal discoidin-like domain. The putative substrate binding and processing site is located on the face of the beta-helix whereas the C-terminal domain is likely involved in carbohydrates binding. NMR spectroscopy and mass spectrometry analyses of degraded LPS (OSA) fragments show an O5 serotype-specific polysaccharide lyase specificity. LKA1gp49 reduces virulence in an in vivo Galleria mellonella infection model and sensitizes P. aeruginosa to serum complement activity. This enzyme causes biofilm degradation and does not affect the activity of ciprofloxacin and gentamicin. This is the first comprehensive report on LPS-degrading lyase derived from a Pseudomonas phage. Biological properties reveal a potential towards its applications in antimicrobial design and as a microbiological or biotechnological tool.
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Bacteriófagos/enzimología , Antígenos O/metabolismo , Polisacárido Liasas/metabolismo , Pseudomonas/virología , Biopelículas , VirulenciaRESUMEN
Vasculogenesis was originally defined by Risau in 1997 [Nature 386: 671-674] as the de novo formation of vessels from endothelial progenitor cells (EPCs), so-called angioblasts. Initially, this process was believed to be related only to embryonic life; however, further studies reported vasculogenesis to occur also in adult tissues. This overview presents the current knowledge about the origin, differentiation and significance of EPCs that have been observed in various diseases, tumors, and reparative processes. We also summarize the knowledge of how to activate these cells for therapeutic purposes and the outcomes of the therapies.
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Tratamiento Basado en Trasplante de Células y Tejidos , Neovascularización Fisiológica , Animales , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Desarrollo Embrionario , Células Progenitoras Endoteliales/citología , HumanosRESUMEN
Angiogenesis contributes to the generation of the vascular bed but also affects the progression of many diseases, such as tumor growth. Many details of the molecular pathways controlling angiogenesis are still undefined due to the lack of appropriate models. We propose the proepicardial explant as a suitable model for studying certain aspects of angiogenesis. The proepicardium (PE) is a transient embryonic structure that contains a population of undifferentiated endothelial cells (ECs) forming a vascular net continuous with the sinus venosus. In this paper, we show that PE explants give rise to CD31-positive vascular sprouts in the presence of basic fibroblast growth factor (bFGF) and 2 isoforms of vascular endothelial growth factor A (VEGF-A), i.e. VEGF-A120 and VEGF-A164. Vascular sprouts exhibit differences in number, length, thickness and the number of branches, depending on the combination of growth factors used. Moreover, the ECs of the sprouts express various levels of mRNA for Notch1 and its ligand Dll4. Additionally, stimulation with bFGF/VEGF-A164 upregulates the expression of Lyve-1 antigen in the ECs in the sprouts. In summary, we present a new model for angiogenesis studies involving mouse PE as a source of ECs. We believe that our model may act as a supplementary assay for angiogenesis studies along with the existing models.
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Inductores de la Angiogénesis/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Pericardio/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacología , Proteínas Adaptadoras Transductoras de Señales , Animales , Biomarcadores/metabolismo , Proteínas de Unión al Calcio , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Pericardio/embriología , Pericardio/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Embarazo , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptor EphB4/genética , Receptor EphB4/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: The proepicardium (PE), a transient embryonic structure crucial for the development of the epicardium and heart, contains its own population of endothelial cells (ECs). The aim of our study was to determine the pattern, anatomical orientation and phenotypic marker expression of the endothelial cell network within the PE. RESULTS: Immunohistochemical findings revealed that proepicardial ECs express both early and late EC-specific markers such as CD31, Flk-1, Lyve-1 and Tie-2 but not SCL/Tal1, vWF, Dll4 or Notch1. Proepicardial ECs are present in the vicinity of the sinus venosus (SV) and form a continuous network of vascular sprouts/tubules connected with the SV endothelium, with Ter-119-positive erythroblasts in the vascular lumina. CONCLUSIONS: On the basis of our results, we postulate the existence of a continuous network of ECs in the PE, exhibiting connection and/or patency with the SV and forming vessels/tubules/strands. Marker expression suggests that ECs are immature and undifferentiated, which was also confirmed with a transmission electron microscopy (TEM) analysis. Our results deliver new data for a better understanding of the nature of proepicardial ECs.
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Células Endoteliales/citología , Células Endoteliales/metabolismo , Pericardio/embriología , Pericardio/metabolismo , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Femenino , Edad Gestacional , Glicoproteínas/metabolismo , Imagenología Tridimensional , Inmunohistoquímica , Masculino , Proteínas de Transporte de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microscopía Electrónica de Transmisión , Modelos Cardiovasculares , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Embarazo , Receptor TIE-2/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.
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Bacteriófagos/fisiología , Biopelículas , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/virología , Bacteriófagos/aislamiento & purificación , Bacteriófagos/ultraestructura , Genoma Viral , Genómica , Filogenia , Receptores ViralesRESUMEN
Vasculogenesis and hematopoiesis are co-localized in the embryonic body, but precise phenotypes of the cells contributing to these processes are not defined. The aim of this study was to characterize phenotypic profiles and location of putative vasculogenic and hematopoietic cellular progenitors in the embryonic mouse heart. Confocal microscopy, as well as ultrastructural and stereomicroscopic analyses, was performed on immunohistochemical whole-mount-stained or sectioned hearts at stages 11.5-14 dpc. A FASC analysis was conducted to quantify putative vasculogenic and hematopoietic cells. We found subepicardial blood islands in the form of foci of accumulation of cells belonging to erythroblastic and megakaryocytic lineages at various stages of maturation, exhibiting phenotypes: GATA2(+)/CD41(+), GATA2(-)/CD41(+), GATA2(+)/CD71(-), GATA2(-)/CD71(+), Fli1(+)/CD71(+), Fli1(-)/CD71(+), with a majority of cells expressing the Ter119 antigen, but none of them expressing Flk1. The subepicardium and the outflow tract endothelium were recognized to be the areas where progenitor cells were scattered or adjoining the endothelial cells. These progenitor cells were characterized as possessing the following antigens: CD45(+)/Fli1(+), CD41(+)/Flk1(+), Flk1(+)/Fli1(+). A FACS analysis demonstrated that the CD41/Flk1 double-positive population of cells constituted 2.68% of total cell population isolated from 12.5 dpc hearts. Vessels and tubules were positive for CD31, Flk1, Fli1, Tie2, including blood islands endothelia. The endocardial wall endothelia were found to function as an anchoring apparatus for megakaryocytes releasing platelets into the cardiac cavities. Phenotypic characteristics of vasculogenic (Flk1(+)/Fli1(+)) and hematopoietic (GATA2(+)/CD71(+), CD41(+)/GATA2(+)) progenitors, as well as the putative hemogenic endothelium (Flk1(+)/CD41(+)) in embryonic mouse hearts, have been presented. Cardiac blood islands, the subepicardium and endothelium of the outflow tract cushions have been defined as areas where these progenitor cells can be found.
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Corazón/embriología , Miocardio/citología , Células Madre/fisiología , Animales , Biomarcadores/metabolismo , Hematopoyesis , Inmunohistoquímica , Antígenos Comunes de Leucocito/metabolismo , Ratones , Microscopía Electrónica de TransmisiónRESUMEN
The adult mouse heart possesses an extensive lymphatic plexus draining predominantly the subepicardium and the outer layer of the myocardial wall. However, the development of this plexus has not been entirely explored, partially because of the lack of suitable methods for its visualization as well as prolonged lymphatic vessel formation that starts prenatally and proceeds during postnatal stages. Also, neither the course nor location of collecting vessels draining lymph from the mouse heart have been precisely characterized. In this article, we report that murine cardiac lymphatic plexus development that is limited prenatally only to the subepicardial area, postnatally proceeds from the subepicardium toward the myocardial wall with the base-to-apex gradient; this plexus eventually reaches the outer half of the myocardium with a predominant location around branches of coronary arteries and veins. Based on multiple marker immunostaining, the molecular marker-phenotype of cardiac lymphatic endothelial cells can be characterized as: Prox-1(+), Lyve-1(+), VEGFR3(+), Podoplanin(+), VEGFR2(+), CD144(+), Tie2(+), CD31(+), vWF(-), CD34(-), CD133(-). There are two major collecting vessels: one draining the right and left ventricles along the left conal vein and running upwards to the left side of the pulmonary trunk and further to the nearest lymph nodes (under the aortic arch and near the trachea), and the other one with its major branch running along the left cardiac vein and further on the surface of the coronary sinus and the left atrium to paratracheal lymph nodes. The extracardiac collectors gain the smooth muscle cell layer during late postnatal stages.
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Células Endoteliales/fisiología , Corazón/anatomía & histología , Linfangiogénesis/fisiología , Vasos Linfáticos/anatomía & histología , Animales , Corazón/crecimiento & desarrollo , RatonesRESUMEN
The interactions between cationic liposomal formulations (PC:Chol:DOTAP 3:4:3) and 23 Pseudomonas aeruginosa strains were tested. The study was undertaken because different antimicrobial results had been obtained by the authors for Pseudomonas aeruginosa strains and liposomal antibiotics (Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz, W., Kozubek, A., 2006. The comparison of in vitro antimicrobial activity of liposomes containing meropenem and gentamicin. Cell. Mol. Biol. Lett., 11, 360-375; Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz W., Kozubek, A., 2006. In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains. Int. J. Pharm., 315, 59-66). The experiments evaluate the roles of the bacterial outer-membrane structure, especially outer-membrane proteins and LPS, and envelope properties (hydrophobicity and electrostatic potential) in the interactions/fusion process between cells and lipid vesicles. The interactions were examined by fluorescent microscopy using PE-rhodamine-labelled liposomes. Some of the strains exhibited red-light emission (fusion with vesicles or vesicles surrounding the cell) and some showed negative reaction (no red-light emission). The main aim of the study was to determine what kinds of bacterial structure or envelope properties have a major influence on the fusion process. Negatively charged cells and hydrophobic properties promote interaction with cationic lipid vesicles, but no specific correlation was noted for the tested strains. A similar situation concerned LPS structure, where parent strains and their mutants possessing identical ladder-like band patterns in SDS-PAGE analysis exhibited totally different results with fluorescent microscopy. Outer-membrane protein analysis showed that an 18-kDA protein occurred in the isolates showing fusion with rhodamine-labelled vesicles and, conversely, strains lacking the 18-kDA protein exhibited no positive reaction (red emission). This suggests that even one protein may be responsible for favouring stronger interactions between Pseudomonas aeruginosa cells and cationic liposomal formulations (PC:Chol:DOTAP 3:4:3).