RESUMEN
The causative agent of Legionnaires' disease, Legionella pneumophila, replicates in free-living amoebae as well as in macrophages of the innate immune system within a distinct membrane-bound compartment, the "Legionella-containing-vacuole" (LCV). LCV formation is a complex process and requires the bacterial Icm/Dot type IV secretion system, which translocates approximately 300 different "effector" proteins. Intact LCVs from infected Dictyostelium discoideum amoebae or RAW 264.7 murine macrophages can be purified using a straightforward protocol. In the first step, the LCVs in cell homogenates are tagged with an antibody directed against an L. pneumophila effector protein specifically localizing to the pathogen vacuole membrane and isolated by immunomagnetic separation using a secondary antibody coupled to magnetic beads. In the second step, the LCVs are further enriched by density gradient centrifugation through a Histodenz cushion. LCVs thus purified are analyzed by mass spectrometry-based proteomics and characterized by biochemical and cell biological approaches.
Asunto(s)
Metabolismo Energético , Interacciones Huésped-Patógeno , Legionella/metabolismo , Proteómica , Proteínas Protozoarias/metabolismo , Vacuolas/metabolismo , Amoeba/metabolismo , Amoeba/microbiología , Animales , Fraccionamiento Celular/métodos , Centrifugación por Gradiente de Densidad , Cromatografía Liquida , Macrófagos/metabolismo , Macrófagos/microbiología , Espectrometría de Masas , Ratones , Fagocitos/inmunología , Fagocitos/metabolismo , Fagocitos/microbiología , Proteómica/métodos , Células RAW 264.7 , Sistemas de Secreción Tipo IVRESUMEN
Legionella pneumophila is an opportunistic bacterial pathogen that causes a severe lung infection termed "Legionnaires' disease." The pathogen replicates in environmental protozoa as well as in macrophages within a unique membrane-bound compartment, the Legionella-containing-vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates ca. 300 "effector proteins" into host cells, where they target distinct host factors. The L. pneumophila "pentuple" mutant (Δpentuple) lacks 5 gene clusters (31% of the effector proteins) and replicates in macrophages but not in Dictyostelium discoideum amoeba. To elucidate the host factors defining a replication-permissive compartment, we compare here the proteomes of intact LCVs isolated from D. discoideum or macrophages infected with Δpentuple or the parental strain Lp02. This analysis revealed that the majority of host proteins are shared in D. discoideum or macrophage LCVs containing the mutant or the parental strain, respectively, whereas some proteins preferentially localize to distinct LCVs. The small GTPase Rap1 was identified on D. discoideum LCVs containing strain Lp02 but not the Δpentuple mutant and on macrophage LCVs containing either strain. The localization pattern of active Rap1 on D. discoideum or macrophage LCVs was confirmed by fluorescence microscopy and imaging flow cytometry, and the depletion of Rap1 by RNA interference significantly reduced the intracellular growth of L. pneumophila Thus, comparative proteomics identified Rap1 as a novel LCV host component implicated in intracellular replication of L. pneumophila.
Asunto(s)
Proteínas Bacterianas/genética , Dictyostelium/metabolismo , Legionella pneumophila/fisiología , Macrófagos/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteómica/métodos , Vacuolas/microbiología , Proteínas de Unión al GTP rap1/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Cromatografía Liquida , Replicación del ADN , Dictyostelium/microbiología , Eliminación de Gen , Legionella pneumophila/genética , Enfermedad de los Legionarios/microbiología , Macrófagos/microbiología , Ratones , Proteínas Protozoarias/metabolismo , Células RAW 264.7 , Espectrometría de Masas en Tándem , Vacuolas/metabolismoRESUMEN
Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.
Asunto(s)
Hidroliasas/inmunología , Interferones/inmunología , Legionella pneumophila/inmunología , Enfermedad de los Legionarios/inmunología , Macrófagos Alveolares/inmunología , Proteoma , Animales , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Ontología de Genes , Hidroliasas/genética , Hidroliasas/metabolismo , Inmunidad Innata , Interferones/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Modelos Inmunológicos , Especies Reactivas de Oxígeno/metabolismo , Succinatos/metabolismo , Vacuolas/metabolismo , Vacuolas/microbiologíaRESUMEN
The causative agent of Legionnaires' disease, Legionella pneumophila, grows in environmental amoebae and mammalian macrophages within a distinct compartment, the 'Legionella-containing vacuole' (LCV). Intracellular bacteria are protected from many antibiotics, and thus are notoriously difficult to eradicate. To identify novel compounds that restrict intracellular bacterial replication, we previously developed an assay based on a coculture of amoebae and GFP-producing L. pneumophila. This assay was used to screen a pathway-based, highly diverse chemical library, referred to as the Sinergia library. In this work, we chose to focus on a group of 11 hit compounds, the majority of which originated from the query molecule CN585, a compound that targets the protein phosphatase calcineurin. Further studies on 78 related compound variants revealed crucial structural attributes, namely a triple-ring scaffold with a central triazine moiety, substituted in positions 3 and 5 by two piperidine or pyrrolidine rings, and in position 1 by an amine group bearing a single aliphatic chain moiety. The most effective compound, ZINC00615682, inhibited intracellular replication of L. pneumophila with an IC50 of approximately 20 nM in Acanthamoeba castellanii and slightly less efficiently in Dictyostelium discoideum or macrophages. Pharmacological and genetic attempts to implicate calcineurin in the intracellular replication of L. pneumophila failed. Taken together, these results show that the amoebae-based screen and structure-activity relationship analysis is suitable for the identification of novel inhibitors of the intracellular replication of L. pneumophila. The most potent compound identified in this study targets (an) as yet unidentified host factor(s).
RESUMEN
Legionella spp. are amoebae-resistant environmental bacteria that replicate in free-living protozoa in a distinct compartment, the Legionella-containing vacuole (LCV). Upon transmission of Legionella pneumophila to the lung, the pathogens employ an evolutionarily conserved mechanism to grow in LCVs within alveolar macrophages, thus triggering a severe pneumonia termed Legionnaires' disease. LCV formation is a complex and robust process, which requires the bacterial Icm/Dot type IV secretion system and involves the amazing number of 300 different translocated effector proteins. LCVs interact with the host cell's endosomal and secretory vesicle trafficking pathway. Accordingly, in a proteomics approach as many as 12 small Rab GTPases implicated in endosomal and secretory vesicle trafficking were identified and validated as LCV components. Moreover, the small GTPase Ran and its effector protein RanBP1 have been found to decorate the pathogen vacuole. Ran regulates nucleo-cytoplasmic transport, spindle assembly, and cytokinesis, as well as the organization of non-centrosomal microtubules. In L. pneumophila-infected amoebae or macrophages, Ran and RanBP1 localize to LCVs, and the small GTPase is activated by the Icm/Dot substrate LegG1. Ran activation by LegG1 leads to microtubule stabilization and promotes intracellular pathogen vacuole motility and bacterial growth, as well as chemotaxis and migration of Legionella-infected cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Legionella pneumophila/fisiología , Vacuolas/fisiología , Proteína de Unión al GTP ran/metabolismo , Amoeba/microbiología , Interacciones Huésped-Patógeno , Legionella pneumophila/enzimología , Legionella pneumophila/patogenicidad , Macrófagos/microbiología , Microtúbulos/metabolismo , Vacuolas/microbiologíaRESUMEN
The opportunistic pathogen Legionella pneumophila employs the Icm/Dot type IV secretion system and â¼300 different effector proteins to replicate in macrophages and amoebae in a distinct 'Legionella-containing vacuole' (LCV). LCVs from infected RAW 264.7 macrophages were enriched by immuno-affinity separation and density gradient centrifugation, using an antibody against the L. pneumophila effector SidC, which specifically binds to the phosphoinositide PtdIns(4)P on the pathogen vacuole membrane. The proteome of purified LCVs was determined by mass spectro-metry (data are available via ProteomeXchange with identifier PXD000647). The proteomics analysis revealed more than 1150 host proteins, including 13 small GTPases of the Rab family. Using fluorescence microscopy, 6 novel Rab proteins were confirmed to localize on pathogen vacuoles harbouring wild-type but not ΔicmT mutant L. pneumophila. Individual depletion of 20 GTPases by RNA interference indicated that endocytic GTPases (Rab5a, Rab14 and Rab21) restrict intracellular growth of L. pneumophila, whereas secretory GTPases (Rab8a, Rab10 and Rab32) implicated in Golgi-endosome trafficking promote bacterial replication. Upon silencing of Rab21 or Rab32, fewer LCVs stained positive for Rab4 or Rab9, implicated in secretory or retrograde trafficking respectively. Moreover, depletion of Rab8a, Rab14 or Rab21 significantly decreased the number of SidC-positive LCVs, suggesting that PtdIns(4)P is reduced under these conditions. L. pneumophila proteins identified in purified LCVs included proteins putatively implicated in phosphorus metabolism and as many as 60 Icm/Dot-translocated effectors, which are likely required early during infection. Taken together, the phagocyte and Legionella proteomes of purified LCVs lay the foundation for further hypothesis-driven investigations of the complex process of pathogen vacuole formation.
Asunto(s)
Legionella pneumophila/fisiología , Proteoma/metabolismo , Vacuolas/enzimología , Proteínas de Unión al GTP rab/fisiología , Animales , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Dictyostelium/fisiología , Interacciones Huésped-Patógeno , Humanos , Macrófagos/enzimología , Macrófagos/microbiología , Ratones , Vacuolas/microbiologíaRESUMEN
CONTEXT: Approaching of pharmaceutical and cosmetic industries in some aspects inevitably influence formulation of topical pharmaceuticals, urging researchers to introduce novel excipients with proven benefits over traditional ones. In that context, alkyl polyglucosides (APG) emerge as prominent natural-origin emulsifiers with numerous favorable features (biodegradability, dermatological acceptability, desirable sensory properties). OBJECTIVE: To evaluate APG-stabilized bases (alone and upon addition of isopropyl alcohol) and their impact on skin performance. A simultaneous in vitro/in vivo skin absorption study was conducted to evaluate whether the tape stripping technique could be recommended as an in vivo tool for skin penetration assessment during formulation optimization process. MATERIALS AND METHODS: After a comprehensive physicochemical characterization, biopharmaceutical properties of APG-bases versus reference ones were assessed through a combined in vitro (release/permeation) and in vivo approach. RESULTS AND DISCUSSION: Physicochemical characterization revealed substantial difference in structural ordering due to the formation of various mesomorphic phases. The enhancer-loaded APG base resulted in significantly higher drug levels at all depths into the stratum corneum, indicating that the selected enhancer along with specific colloidal structure has increased the extent of drug delivery. CONCLUSION: Results recommend the investigated emulsifier for stabilization of topical drug delivery systems, not only for their ability to sustain the addition of isopropyl alcohol which proved to be a valuable enhancer, but also satisfactory skin absorption and tolerability when compared to samples stabilized by conventional emulsifier. Tape stripping proved to be a useful and yet inexpensive tool for in vivo trials, able to discriminate subtle differences in dermal availability.
Asunto(s)
2-Propanol/química , Composición de Medicamentos/métodos , Emulsionantes/química , Alcoholes Grasos/química , Glucolípidos/química , Piel/metabolismo , Rastreo Diferencial de Calorimetría , Diclofenaco/administración & dosificación , Diclofenaco/química , Diclofenaco/farmacocinética , Liberación de Fármacos , Femenino , Humanos , Técnicas In Vitro , Reología , Absorción Cutánea , Solubilidad , TermogravimetríaRESUMEN
The severe pneumonia known as Legionnaires' disease occurs following infection by the Gram-negative bacterium Legionella pneumophila. Normally resident in fresh-water sources, Legionella are subject to predation by eukaryotic phagocytes such as amoeba and ciliates. To counter this, L. pneumophila has evolved a complex system of effector proteins which allow the bacteria to hijack the phagocytic vacuole, hiding and replicating within their erstwhile killers. These same mechanisms allow L. pneumophila to hijack another phagocyte, lung-based macrophages, which thus avoids a vital part of the immune system and leads to infection. The course of infection can be divided into five main categories: pathogen uptake, formation of the replication-permissive vacuole, intracellular replication, host cell response, and bacterial exit. L. pneumophila effector proteins target every stage of this process, interacting with secretory, endosomal, lysosomal, retrograde and autophagy pathways, as well as with mitochondria. Each of these steps can be studied in protozoa or mammalian cells, and the knowledge gained can be readily applied to human pathogenicity. Here we describe the manner whereby L. pneumophila infects host protozoa, the various techniques which are available to analyse these processes and the implications of this model for Legionella virulence and the pathogenesis of Legionnaires' disease.
Asunto(s)
Alveolados , Células Eucariotas/microbiología , Interacciones Huésped-Patógeno , Legionella pneumophila/fisiología , Enfermedad de los Legionarios , Animales , Humanos , Modelos TeóricosRESUMEN
The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct "Legionella-containing vacuole" (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Legionella pneumophila/fisiología , Macrófagos/microbiología , Microtúbulos/metabolismo , Fagosomas/metabolismo , Proteína de Unión al GTP ran/metabolismo , Animales , Proteínas Bacterianas/genética , Línea Celular , Activación Enzimática , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Proteínas Activadoras de GTPasa/genética , Silenciador del Gen , Humanos , Legionella pneumophila/genética , Legionella pneumophila/inmunología , Legionella pneumophila/ultraestructura , Enfermedad de los Legionarios/inmunología , Enfermedad de los Legionarios/metabolismo , Enfermedad de los Legionarios/microbiología , Enfermedad de los Legionarios/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/ultraestructura , Ratones , Proteínas de Microtúbulos/química , Proteínas de Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mutación , Fagocitosis , Fagosomas/enzimología , Fagosomas/ultraestructura , Polimerizacion , Estabilidad Proteica , Transporte de Proteínas , Replicación Viral , Proteína de Unión al GTP ran/antagonistas & inhibidores , Proteína de Unión al GTP ran/genéticaRESUMEN
The bacteria causing Legionnaires' disease, Legionella pneumophila, replicate intracellularly within unique Legionella-containing vacuoles (LCVs). LCV formation involves a type IV secretion system (T4SS) that translocates effector proteins into host cells. We show that the T4SS effector RidL localizes to LCVs, supports intracellular bacterial growth, and alters retrograde trafficking, in which selected proteins are transported from endosomes to the Golgi. The retromer complex that mediates retrograde trafficking localizes to LCVs independently of RidL and restricts intracellular bacterial growth. RidL binds the Vps29 retromer subunit and the lipid PtdIns(3)P, which localizes retromer components to membranes. Additionally, specific retromer cargo receptors and sorting nexins that mediate protein capture and membrane remodeling preferentially localize to LCVs in the absence of ridL. Ectopic RidL production inhibits retrograde trafficking, and L. pneumophila blocks retrograde transport at endosome exit sites in a ridL-dependent manner. Collectively, these findings suggest that RidL inhibits retromer function to promote intracellular bacterial replication.
Asunto(s)
Proteínas Bacterianas/metabolismo , Legionella pneumophila/metabolismo , Enfermedad de los Legionarios/metabolismo , Enfermedad de los Legionarios/microbiología , Vacuolas/microbiología , Animales , Proteínas Bacterianas/genética , Línea Celular , Endosomas/metabolismo , Humanos , Legionella pneumophila/genética , Ratones , Transporte de Proteínas , Vacuolas/metabolismoRESUMEN
Protozoa are natural reservoirs of the environmental bacterium Legionella pneumophila. Upon inhalation of Legionella-laden aerosols, the amoeba-resistant bacteria replicate within human alveolar macrophages causing the severe pneumonia "Legionnaires' disease." Within host cells, including Dictyostelium discoideum, L. pneumophila establishes a custom-tailored compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system and involves a plethora of "effector" proteins, some of which specifically decorate the LCV membrane. This unique feature of LCVs is exploited to isolate the pathogen vacuole by immunomagnetic separation using an antibody against the effector protein SidC. LCV purity is further increased by a subsequent density gradient centrifugation step. The use of red fluorescent L. pneumophila and D. discoideum producing the LCV marker calnexin-GFP allows following the purification by fluorescence microscopy.
Asunto(s)
Fraccionamiento Celular/métodos , Dictyostelium/microbiología , Legionella pneumophila/fisiología , Vacuolas/microbiología , Centrifugación por Gradiente de Densidad , Técnicas de Cultivo , Interacciones Huésped-Patógeno , Humanos , Separación Inmunomagnética/métodos , Microscopía FluorescenteRESUMEN
Legionella pneumophila replicates intracellularly in environmental and immune phagocytes within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is strictly dependent on the Icm/Dot type IV secretion system and the translocation of "effector" proteins into the cell. Some effector proteins decorate the LCV membrane and subvert host cell vesicle trafficking pathways. Here we describe a method to purify intact LCVs from Dictyostelium discoideum amoebae and RAW 264.7 murine macrophages. The method comprises a two-step protocol: first, LCVs are enriched by immuno-magnetic separation using an antibody against a bacterial effector protein specifically localizing to the LCV membrane, and second, the LCVs are further purified by density gradient centrifugation. The purified LCVs can be characterized by proteomics and other biochemical approaches.
Asunto(s)
Amoeba/microbiología , Fraccionamiento Celular/métodos , Legionella/fisiología , Macrófagos/microbiología , Vacuolas/microbiología , Animales , Línea Celular , Centrifugación por Gradiente de Densidad , Legionella pneumophila/fisiología , Ratones , Microscopía FluorescenteRESUMEN
The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"(1). In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles(2-4). The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics. The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania(5), Listeria(6), Mycobacterium(7), Rhodococcus(8), Salmonella(9) or Legionella spp.(10). However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment. The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)(3,11) . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation(12,13) (Fig. 1). A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before(13). LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.
Asunto(s)
Dictyostelium/microbiología , Legionella pneumophila/aislamiento & purificación , Fagocitos/microbiología , Vacuolas/microbiología , Animales , Western Blotting , Dictyostelium/química , Dictyostelium/aislamiento & purificación , Legionella pneumophila/química , Macrófagos/química , Macrófagos/microbiología , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Fagocitos/química , Proteómica , Vacuolas/químicaRESUMEN
The ViSC Social Competence Program has been implemented in Austrian schools within the scope of a national strategy plan, Together Against Violence. The program is a primary preventive program designed for grades 5 to 8. The prevention of aggression and bullying is defined as a school development task, and the initial implementation of the program lasts one school year. The program consists of universal and specific actions that are implemented through in-school teacher training and a class project for students. The program was evaluated with a randomized intervention control group design. Data were collected from teachers and students. Results suggest that the program reduces aggression in schools.
Asunto(s)
Acoso Escolar , Evaluación de Programas y Proyectos de Salud , Servicios de Salud Escolar/organización & administración , Conducta Social , Estudiantes/psicología , Violencia/prevención & control , Agresión , Austria , Niño , Docentes , Femenino , Humanos , Masculino , Servicios de Salud Escolar/normas , Estudiantes/estadística & datos numéricos , Enseñanza/métodosRESUMEN
Recently, healthcare professionals again began realizing the benefits of preparing customized medications to meet specific patient needs. The objective of this work was to develop and evaluate simple pharmaceutical bases stabilized with natural-origin surfactant of alkyl polyglucoside (APG) type as prospective ready-to-use bases and compare them to widely used pharmacopoeial ones. Additionally, the ability of the formulated bases to sustain isopropyl alcohol was assessed as well as its influence on ketoprofen skin absorption (as a co-solvent and potential penetration enhancer). In order to evaluate the manifold characteristics a topical drug product should possess, a comprehensive characterization was performed using different techniques. Physicochemical characterization demonstrated satisfactory physical stability of APG-stabilized bases upon the addition of alcohol. In vitro release/permeation studies failed to show significant difference in ketoprofen liberation/permeation profiles from different bases. However, the extent of ketoprofen delivery in vivo was clearly increased from APG bases, relative to that obtained from pharmacopoeia quality one, implying a distinct influence of the emulsion systems' colloidal structures. Taking also into account the rheological behavior of APG bases, revealing their ameliorated sensory characteristics, it could be concluded that the investigated APG bases could be considered as preferential option in drug compounding related to the conventional ones.
Asunto(s)
Cetoprofeno/química , Cetoprofeno/farmacología , Piel/efectos de los fármacos , Piel/metabolismo , Tensoactivos/química , 2-Propanol/química , Administración Tópica , Adulto , Química Farmacéutica/métodos , Coloides/química , Coloides/farmacología , Composición de Medicamentos/métodos , Emulsiones/química , Emulsiones/farmacología , Femenino , Humanos , Cetoprofeno/farmacocinética , Masculino , Permeabilidad , Estudios Prospectivos , Reología/métodos , Absorción Cutánea , Piel Artificial , Solventes/química , Tensoactivos/farmacocinética , Tensoactivos/farmacología , Adulto JovenRESUMEN
Invasion of mammalian cells via cell adhesion molecules of the integrin family is a common theme in bacterial pathogenesis. Whereas some microorganisms directly bind to integrins, other pathogens such as Staphylococcus aureus indirectly engage these receptors via fibronectin-binding proteins (FnBPs). In this review, we summarize the structure-function relationship of FnBPs and the current view of the role of these proteins during pathogenesis in vivo. A major focus will be on recent findings on the role of cholesterol- and sphingolipid-rich membrane microdomains for integrin-initiated uptake of fibronectin-binding bacteria and the surprising inhibitory function of caveolin-1 in this process. The detailed mechanistic understanding of host cell invasion by fibronectin-binding S. aureus can not only serve as a paradigm for other fibronectin-binding pathogenic bacteria, but might also reveal the physiological regulation of endocytosis of ligand-occupied integrins.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Bacterias/metabolismo , Bacterias/patogenicidad , Endocitosis , Integrinas/metabolismo , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Adhesinas Bacterianas/química , Animales , Adhesión Bacteriana , Humanos , Microdominios de Membrana , Ratones , Staphylococcus aureus/citología , Relación Estructura-ActividadRESUMEN
To establish bovine spongiform encephalopathy (BSE) public health protection measures it is important to precisely define the cattle tissues considered as specified risk materials (SRM). To date, in pre-clinical BSE infected cattle, no evidence of the BSE agent had been found in the gut outside of the ileal Peyer's Patches. This study was undertaken to determine when and where the pathological prion protein (PrPSc) and/or BSE infectivity can be found in the small intestine of cattle 4 to 6 months of age, orally challenged with BSE. Samples of the jejunum, the ileum and the ileocaecal junction from 46 BSE infected cattle, culled from 1 up to 44 months post infection (mpi) were examined by immunohistochemistry. Samples from cattle 8 mpi to 20 mpi were additionally studied by PTA Western blot, rapid tests, and by mouse (TgbovXV) bioassay. In doing so nearly all of the cattle, from 4 up to 44 mpi, had detectable amounts of PrPSc and/or infectivity in the distal ileum. In the distal ileum clear time-dependent variations were visible concerning the amount of PrPSc, the tissue structures affected, and the cells involved. BSE infectivity was found not only in the ileum and ileocaecal junction but also in the jejunum. The systematic approach of this study provides new data for qualitative and quantitative risk assessments and allows defining bovine SRM more precisely.
Asunto(s)
Encefalopatía Espongiforme Bovina/patología , Íleon/patología , Yeyuno/patología , Proteínas PrPSc/análisis , Envejecimiento , Animales , Western Blotting/veterinaria , Bovinos , Encefalopatía Espongiforme Bovina/transmisión , Femenino , Ratones , Ratones Transgénicos , Medición de RiesgoRESUMEN
The pathogenesis of bovine spongiform encephalopathy (BSE) infections in cattle has been studied in recent years by using highly sensitive transgenic-mouse bioassays. It has been shown that in this species, the BSE agent amplifies almost exclusively in the central and peripheral nervous system. Even in animals that were killed in the clinical end stage of the disease, the lymphoreticular system was shown to be free of the infectious agent. No other animal species investigated to date exhibits such a restricted BSE-infectivity distribution pattern. However, there is growing evidence for a radial spread of infection from the central nervous system (CNS) into the periphery during the late stages of the disease. In this study, we challenged transgenic mice overexpressing the bovine prion protein with homogenates prepared from a wide variety of tissue samples collected from BSE-infected cattle. As prion infections involve the conversion of the cellular prion protein into its abnormally folded isoform (PrP(Sc)), we applied various detection methods, such as the purification of scrapie-associated fibrils, immunohistochemistry, and the protein misfolding cyclic amplification technique. Despite negative results using these highly sensitive biochemical methods, we were, for the first time, able to detect BSE infectivity in the tongue and in the nasal mucosa of terminally diseased BSE field cases as well as experimentally challenged cattle by transgenic-mouse bioassay. This shows that BSE infectivity can be present in the peripheral tissues of terminally diseased cattle, including tissues used for human consumption.
Asunto(s)
Encefalopatía Espongiforme Bovina/transmisión , Mucosa Nasal/metabolismo , Proteínas PrPSc/metabolismo , Lengua/metabolismo , Animales , Bioensayo , Bovinos , Humanos , Ratones , Ratones Transgénicos , Sensibilidad y EspecificidadRESUMEN
Passiflora incarnata L. (Passifloraceae) is important in herbal medicine for treating anxiety or nervousness, Generalized Anxiety Disorder (GAD), symptoms of opiate withdrawal, insomnia, neuralgia, convulsion, spasmodic asthma, ADHD, palpitations, cardiac rhythm abnormalities, hypertension, sexual dysfunction and menopause. However, the mechanism of action is still under discussion. Despite gaps in our understanding of neurophysiological processes, it is increasingly being recognized that dysfunction of the GABA system is implicated in many neuropsychiatric conditions, including anxiety and depressive disorders. Therefore, the in vitro effects of a dry extract of Passiflora incarnata (sole active ingredient in Pascoflair® 425 mg) on the GABA system were investigated. The extract inhibited [(3) H]-GABA uptake into rat cortical synaptosomes but had no effect on GABA release and GABA transaminase activity. Passiflora incarnata inhibited concentration dependently the binding of [(3) H]- SR95531 to GABA(A) -receptors and of [(3) H]-CGP 54626 to GABA(B) -receptors. Using the [(35) S]-GTPγS binding assay Passiflora could be classified as an antagonist of the GABA(B) receptor. In contrast, the ethanol- and the benzodiazepine-site of the GABA(A) -receptor were not affected by this extract. In conclusion, the first evidence was shown that numerous pharmacological effects of Passiflora incarnata are mediated via modulation of the GABA system including affinity to GABA(A) and GABA(B) receptors, and effects on GABA uptake.
Asunto(s)
Antagonistas del GABA/farmacología , Passiflora/química , Extractos Vegetales/farmacología , Ácido gamma-Aminobutírico/metabolismo , 4-Aminobutirato Transaminasa/metabolismo , Animales , Ansiedad/tratamiento farmacológico , Ansiedad/metabolismo , Unión Competitiva , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Depresión/tratamiento farmacológico , Depresión/metabolismo , Masculino , Plantas Medicinales/química , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Bacteria of the genus Legionella persist in a wide range of environmental habitats, including biofilms, protozoa and nematodes. Legionellaceae are 'accidental' human pathogens that upon inhalation cause a severe pneumonia termed 'Legionnaires' disease'. The interactions of L. pneumophila with eukaryotic hosts are governed by the Icm/Dot type IV secretion system (T4SS) and more than 150 'effector proteins', which subvert signal transduction pathways and promote the formation of the replication-permissive 'Legionella-containing vacuole'. The Icm/Dot T4SS is essential to infect free-living protozoa, such as the amoeba Dictyostelium discoideum, as well as the nematode Caenorhabditis elegans, or mammalian macrophages. To adapt to different niches, L. pneumophila not only responds to exogenous cues, but also to endogenous signals, such as the α-hydroxyketone compound LAI-1 (Legionella autoinducer-1). The long-term adaptation of Legionella spp. is based on extensive horizontal DNA transfer. In fact, Legionella spp. have acquired canonical 'genomic islands' of prokaryotic origin, but also a number of eukaryotic genes. Since many aspects of Legionella virulence against environmental predators and immune phagocytes are similar, an understanding of Legionella ecology provides valuable insights into the pathogenesis of legionellaceae for humans.