RESUMEN
Regulation of cytotoxic effector molecule expression in human CTLs after viral or bacterial activation is poorly understood. By using human autologous dendritic cells (DCs) to prime T lymphocytes, we found perforin only highly up-regulated in virus- (HSV-1, vaccinia virus) but not in intracellular bacteria- (Listeria innocua, Listeria monocytogenes, Mycobacterium tuberculosis, Chlamydophila pneumoniae) activated CTLs. In contrast, larger quantities of IFN-gamma and TNF-alpha were produced in Listeria-stimulated cultures. Granzyme B and granulysin were similarly up-regulated by all tested viruses and intracellular bacteria. DCs infected with HSV-1 showed enhanced surface expression of the costimulatory molecule CD252 (CD134L) compared with Listeria-infected DC and induced enhanced secretion of IL-2. Adding blocking CD134 or neutralizing IL-2 Abs during T cell activation reduced the HSV-dependent up-regulation of perforin. These data indicate a distinct CTL effector function in response to intracellular pathogens triggered via differing endogenous IL-2 production upon costimulation through CD252.
Asunto(s)
Citotoxicidad Inmunológica/inmunología , Interleucina-2/inmunología , Activación de Linfocitos/inmunología , Ligando OX40/inmunología , Linfocitos T/inmunología , Células Cultivadas , Chlamydophila/inmunología , Células Dendríticas/inmunología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/patogenicidad , Humanos , Interleucina-2/metabolismo , Listeria/inmunología , Listeria/patogenicidad , Mycobacterium tuberculosis/inmunología , Perforina/inmunología , Linfocitos T/metabolismo , Transcripción Genética/genética , Regulación hacia Arriba/inmunología , Virus Vaccinia/inmunologíaRESUMEN
BACKGROUND: Atherosclerosis is still the leading cause of death in the western world. Besides known risk factors studies demonstrating Chlamydophila pneumoniae (C. pneumoniae) to be implicated in the progression of the disease, little is known about C. pneumoniae infection dynamics. We investigated whether C. pneumoniae induce cell death of human aortic endothelial cells, a cell type involved in the initiation of atherosclerosis, and whether chlamydial spots derive from inclusions. RESULTS: Lactate dehydrogenase release revealed host cell death to be dependent on the amounts of Chlamydia used for infection. The morphology of lysed human aortic endothelial cells showed DNA strand breaks simultaneously with cell membrane damage exclusively in cells carrying Chlamydia as spots. Further ultrastructural analysis revealed additional organelle dilation, leading to the definition as aponecrotic cell death of endothelial cells. Exclusive staining of the metabolic active pathogens by chlamydial heat shock protein 60 labelling and ceramide incorporation demonstrated that the bacteria responsible for the induction of aponecrosis had resided in former inclusions. Furthermore, a strong pro-inflammatory molecule, high mobility group box protein 1, was shown to be released from aponecrotic host cells. CONCLUSION: From the data it can be concluded that aponecrosis inducing C. pneumoniae stem from inclusions, since metabolically active bacterial spots are strongly associated with aponecrosis late in the infectious cycle in vascular endothelial cells and metabolic activity was exclusively located inside of inclusions in intact cells. Vice versa initial spot-like infection with metabolically inert bacteria does not have an effect on cell death induction. Hence, C. pneumoniae infection can contribute to atherosclerosis by initial endothelial damage.
Asunto(s)
Aterosclerosis/microbiología , Infecciones por Chlamydophila/microbiología , Chlamydophila pneumoniae , Cuerpos de Inclusión/microbiología , Aorta , Apoptosis , Proteínas Bacterianas/metabolismo , Línea Celular , Chaperonina 60/metabolismo , Infecciones por Chlamydophila/patología , Chlamydophila pneumoniae/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Células Endoteliales/patología , Proteína HMGB1/metabolismo , Humanos , Necrosis/patologíaRESUMEN
BACKGROUND: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an essential role in the host defence against intracellular pathogens such as Listeria, and Mycobacteria. The key mediator of bacteria-directed cytotoxicity is granulysin, a 9 kDa protein stored in cytolytic granules together with perforin and granzymes. Granulysin binds to cell membranes and is subsequently taken up via a lipid raft-associated mechanism. In dendritic cells (DC) granulysin is further transferred via early endosomes to L. innocua-containing phagosomes were bacteriolysis is induced. In the present study we analysed the role of perforin in granulysin-induced intracellular bacteriolysis in DC. RESULTS: We found granulysin-induced lysis of intracellular Listeria significantly increased when perforin was simultaneously present. In pulse-chase experiments enhanced bacteriolysis was observed when perforin was added up to 25 minutes after loading the cells with granulysin demonstrating no ultimate need for simultaneous uptake of granulysin and perforin. The perforin concentration sufficient to enhance granulysin-induced intracellular bacteriolysis did not cause permanent membrane pores in Listeria-challenged DC as shown by dye exclusion test and LDH release. This was in contrast to non challenged DC that were more susceptible to perforin lysis. For Listeria-challenged DC, there was clear evidence for an Ca2+ influx in response to sublytic perforin demonstrating a short-lived change in the plasma membrane permeability. Perforin treatment did not affect granulysin binding, initial uptake or intracellular trafficking to early endosomes. However, enhanced colocalization of granulysin with listerial DNA in presence of perforin was found by confocal laser scanning microscopy. CONCLUSION: The results provide evidence that perforin increases granulysin-mediated killing of intracellular Listeria by enhanced phagosome-endosome fusion triggered by a transient Ca2+ flux.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Bacteriólisis/fisiología , Citotoxicidad Inmunológica/fisiología , Células Dendríticas/fisiología , Listeria/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Antígenos de Diferenciación de Linfocitos T/farmacología , Bacteriólisis/efectos de los fármacos , Calcio/fisiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Citotoxicidad Inmunológica/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/microbiología , Endosomas/efectos de los fármacos , Endosomas/microbiología , Endosomas/fisiología , Humanos , Glicoproteínas de Membrana/farmacología , Microdominios de Membrana , Viabilidad Microbiana , Perforina , Proteínas Citotóxicas Formadoras de Poros/farmacología , Proteínas Recombinantes/farmacologíaRESUMEN
BACKGROUND: Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. RESULTS: In this study we investigated generation of granulysin in lymphokine activated killer (LAK) cells and antigen (Listeria) specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation. Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. CONCLUSION: This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/metabolismo , Regulación de la Expresión Génica , Células Asesinas Activadas por Linfocinas/inmunología , Activación de Linfocitos , Procesamiento Proteico-Postraduccional , Linfocitos T/inmunología , Epítopos/inmunología , Humanos , Células Asesinas Activadas por Linfocinas/citología , Cinética , Listeria/inmunología , Microscopía Confocal , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T/citología , Factores de Tiempo , Transcripción GenéticaRESUMEN
BACKGROUND: Silicone oil endotamponade is used for the repair of complicated retinal detachments. Cataract, glaucoma and corneal endothelial dysfunction are the most frequent complications of silicone oil tamponade. Clinical and histopathological studies have revealed that silicone oil can penetrate into the optic nerve and into the brain. The mechanism by which silicone oil moves from intraocular into the optic nerve is still under debate. To investigate the effect of intraocular pressure only, a post-mortem experimental histological study was performed to determine whether silicone oil penetration from the globe into the optic nerve after vitrectomy and silicone oil instillation is a purely pressure-related phenomenon. Although a post-mortem study excludes physiological processes, it serves as a model for the study of pure physical forces onto biological structures. METHODS: The study was carried out on 20 human eyes with their optic nerves attached. All specimens had been harvested from patients without known eye disease. The vitreous body was removed with a syringe and the globe was filled with silicone oil. A lipophil fluorescence marker (Bodipy) was added in 8 eyes. The mean intraocular pressure after silicone oil filling measured 40 mm Hg and the globes stayed under pressure for up to 16 weeks. The eyes and optic nerves were stained with H&E and examined with light, phase-contrast and fluorescence microscopy. RESULTS: None of the 20 specimens examined showed silicone oil in the retrolaminar portion of the optic nerve. CONCLUSIONS: Migration of silicone oil into the optic nerve was not demonstrated in this human post-mortem study. Therefore other factors, such as pre-existing glaucomatous damage to the disc region and/or active transport mechanisms must be involved in the development of silicone oil-associated optic neuropathy.
Asunto(s)
Presión Intraocular , Enfermedades del Nervio Óptico/inducido químicamente , Aceites de Silicona/efectos adversos , Anciano , Cadáver , Femenino , Humanos , Masculino , Microscopía Fluorescente/métodos , Modelos Neurológicos , Nervio Óptico/efectos de los fármacos , Nervio Óptico/patología , Enfermedades del Nervio Óptico/patología , Enfermedades del Nervio Óptico/fisiopatología , Retina/efectos de los fármacos , Retina/patología , Desprendimiento de Retina/patologíaRESUMEN
OBJECTIVES: To assess the suitability of a new anatomic model of the paranasal sinuses for endonasal surgical training. STUDY DESIGN: Prospective observational pilot study. METHODS: A new anatomic model of the paranasal sinuses was developed by the Department of Anatomy at the University of Zurich. The practicability of the model was evaluated by three experienced endoscopic sinus surgeons with a special focus on its possible use in training. Standardized surgical procedures were performed under simulated real-life conditions in the operating theatre. RESULTS: The endoscopic appearance of the nasal airway closely resembled real human tissue and the detailed anatomy of the model allowed the same structured surgical steps to be performed as in real life in the absence of bleeding. CONCLUSION: This anatomic model is a readily available teaching tool for endoscopic sinus surgeons.
Asunto(s)
Endoscopía/educación , Modelos Anatómicos , Nariz/anatomía & histología , Senos Paranasales/anatomía & histología , Anciano , Cadáver , Senos Etmoidales/anatomía & histología , Senos Etmoidales/cirugía , Seno Frontal/anatomía & histología , Seno Frontal/cirugía , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Seno Maxilar/anatomía & histología , Seno Maxilar/cirugía , Nariz/cirugía , Otolaringología/educación , Senos Paranasales/cirugía , Proyectos Piloto , Estudios Prospectivos , Seno Esfenoidal/anatomía & histología , Seno Esfenoidal/cirugía , Materiales de Enseñanza , Grabación en VideoRESUMEN
A multitude of various materials are available for the visualization of cadaveric vessels, ranging from natural materials like gelatin and latex to synthetic materials like silicone rubber or acrylates. To achieve a detailed overview of the vascular architecture in microvascular studies in experimental flap surgery, the injected material should have low viscosity to assure perfusion of even the smallest vessels. In addition, the material ideally should have either no or only minimal shrinkage, and should harden within a reasonable time, but retain sufficient elasticity and resistance to withstand tearing off the delicate vessels during subsequent dissection or casting. Because none of the available injection materials adequately combines these attributes, we evaluated the polyurethane elastomer "PU4ii" in latissimus dorsi muscles as a new material for the visualization of cadaveric vessels in comparison with the frequently used silicone rubber. The dissection of vessels injected with PU4ii proved easy largely because of its exceptional hardness. Even if not visible before dissection, the completely perfused vessels were easily palpated in the surrounding fat or muscle tissue of the microsurgical latissimus dorsi model. Despite the significantly higher hardness of PU4ii over silicone rubber (98 Sh-A vs. 12 Sh-A), PU4ii proved enough elasticity (20-25 N/mm(2) E modulus) and a high tear resistance (64-68 N/mm vs. 15 N/mm) preventing breakage during dissection even within the smallest vessels. In contrast to silicone rubber (and latex or gelatin), the high corrosion resistance and form stability of PU4ii also allowed building of casts for qualitative examination by scanning electron microscopy and quantitative analysis of the vessel density using micro-computed tomography with accurate 3D representation. In this study we show that PU4ii has physical characteristics that make it a multi-purpose material that allows at the same breath an excellent gross visualization of the architecture of cadaveric blood vessels as well as a detailed evaluation of casts by modern microscopic and or radiologic tools. Thus, the new polyurethane elastomer PU4ii is in many respects superior to the widely used silicone rubber and can be strongly recommended as a visualization material for a comprehensive evaluation of cadaveric blood vessels in microsurgery.
Asunto(s)
Vasos Sanguíneos/anatomía & histología , Elastómeros , Embalsamiento/métodos , Poliuretanos , Anciano , Anciano de 80 o más Años , Vasos Sanguíneos/citología , Vasos Sanguíneos/ultraestructura , Cadáver , Molde por Corrosión , Femenino , Humanos , Masculino , Ensayo de Materiales , Músculo Esquelético/irrigación sanguínea , Elastómeros de Silicona , Colgajos QuirúrgicosRESUMEN
BACKGROUND: This study was performed to determine the variations in the branching pattern of the sphenopalatine artery medial to the crista ethmoidalis. Seventy-seven cadaver head sides that had been sectioned sagittally in the midline with their septum removed were used after injecting pink latex to highlight the arterial vessels. METHODS: The mucosa from the middle meatus from the level of the basal lamella was removed until the artery and its branches were seen and then was examined under the microscope to identify the position of the arterial branches. RESULTS: The sphenopalatine artery and its branches were identified in 75 specimens. Of these 75 specimens, 73 (97%) had 2 or more branches medial to the crista ethmoidalis, 49 (67%) had 3 or more branches, 26 (35%) had 4 or more branches, and 1 specimen had 10 branches. In two specimens the artery presented as a single trunk. CONCLUSION: The sphenopalatine artery normally starts to branch lateral to the crista ethmoidalis and these branches vary widely. It is important that the surgeon who undertakes ligation or cautery of the artery is aware of these variations, otherwise they may overlook some of the branches. With an endoscopic approach, removal of the crista ethmoidalis helps visualize these branches.
Asunto(s)
Endoscopía , Seno Esfenoidal/irrigación sanguínea , Cadáver , Humanos , Seno Esfenoidal/anatomía & histologíaRESUMEN
Accumulation of the beta-amyloid peptide (Abeta) in the brain is a major pathological hallmark of Alzheimer's disease (AD), leading to synaptic dysfunction, neuronal death, and memory impairment. The levels of neprilysin, a major Abeta-degrading enzyme, are decreased in AD brains and during aging. Because neprilysin cleaves Abeta in vivo, its down-regulation may contribute to the pathophysiology of AD. The aim of this study was to assess the consequences of neprilysin deficiency on accumulation of murine Abeta in brains and associated pathologies in vivo by investigating neprilysin-deficient mice on biochemical, morphological, and behavioral levels. Aged neprilysin-deficient mice expressed physiological amyloid precursor protein (APP) levels and exhibited elevated brain Abeta concentrations and amyloid-like deposits in addition to signs of neuronal degeneration in their brains. Behaviorally, neprilysin-deficient mice acquired a significantly weaker conditioned taste aversion that extinguished faster than the aversion of age-matched controls. Our data establish that, under physiological APP expression levels, neprilysin deficiency is associated with increased Abeta accumulation in the brain and leads to deposition of amyloid-like structures in vivo as well as with signs of AD-like pathology and with behavioral deficits.
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Amiloide/metabolismo , Conducta Animal/fisiología , Encéfalo/patología , Neprilisina/deficiencia , Factores de Edad , Amiloide/ultraestructura , Análisis de Varianza , Animales , Reacción de Prevención/fisiología , Encéfalo/ultraestructura , Condicionamiento Operante/fisiología , Ensayo de Inmunoadsorción Enzimática/métodos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión/métodos , Privación de Agua/fisiologíaRESUMEN
The release of granulysin, a 9-kDa cationic protein, from lysosomal granules of cytotoxic T lymphocytes and natural killer cells plays an important role in host defense against microbial pathogens. Granulysin is endocytosed by the infected target cell via lipid rafts and kills subsequently intracellular bacteria. The mechanism by which granulysin binds to eukaryotic and prokaryotic cells but lyses only the latter is not well understood. We have studied the effect of granulysin on large unilamellar vesicles (LUVs) and supported bilayers with prokaryotic and eukaryotic lipid mixtures or model membranes with various lipid compositions and charges. Binding of granulysin to bilayers with negative charges, as typically found in bacteria and lipid rafts of eukaryotic cells, was shown by immunoblotting. Fluorescence release assays using LUV revealed an increase in permeability of prokaryotic, negatively charged and lipid raft-like bilayers devoid of cholesterol. Changes in permeability of these bilayers could be correlated to defects of various sizes penetrating supported bilayers as shown by atomic force microscopy. Based on these results, we conclude that granulysin causes defects in negatively charged cholesterol-free membranes, a membrane composition typically found in bacteria. In contrast, granulysin is able to bind to lipid rafts in eukaryotic cell membranes, where it is taken up by the endocytotic pathway, leaving the cell intact.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/metabolismo , Colesterol/fisiología , Membrana Dobles de Lípidos/metabolismo , Antígenos de Diferenciación de Linfocitos T/farmacología , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Humanos , Membrana Dobles de Lípidos/química , Permeabilidad/efectos de los fármacos , Electricidad EstáticaRESUMEN
The role of human dendritic cells (DC) in the immune response toward intracellularly growing Listeria was analyzed under in vitro conditions using several morphological and functional methods. DC incubated with Listeria innocua and L. monocytogenes, respectively, readily phagocytosed the bacteria. Listeria did not impair viability and immunogenic potential of human DC. Listerial antigens were found to be processed within the lysosomal compartment of DC and colocalized with major histocompatibility complex (MHC) class II molecules, as shown by fluorescence and transmission electron microscopy. DC challenged with apathogenic L. innocua were highly effective in priming autologous naive T cells (mainly CD4+) in vitro. The T cells strongly proliferated in the presence of DC incubated with L. innocua, which could be significantly inhibited by anti-MHC II mAb. L. innocua-primed T cells were also successfully stimulated by DC harboring the pathogenic L. monocytogenes, either the wild-type strain EGD or the p60 reduced mutant strain RIII. From our results, we conclude that human DC infected with nonpathogenic intracellular bacteria are able to efficiently prime naive T cells, which are then suitable for recognition of antigens derived from related virulent bacterial species. This in vitro human model provides an interesting tool for basic research in infectious immunology and possibly for a new immunotherapy.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Listeria/inmunología , Antígenos CD/inmunología , Antígeno B7-2 , Proliferación Celular , Supervivencia Celular/inmunología , Células Dendríticas/microbiología , Células Dendríticas/ultraestructura , Citometría de Flujo , Gentamicinas/farmacología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunoglobulinas/inmunología , Listeria/efectos de los fármacos , Listeria/ultraestructura , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/inmunología , Listeria monocytogenes/ultraestructura , Glicoproteínas de Membrana/inmunología , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Antígeno CD83RESUMEN
A variety of explanations have been provided to elucidate the requirement of the large islet mass that is essential for a successful treatment of patients with type I diabetes by intrahepatic transplantation. The purpose of this study was to investigate islet cell survival under the effect of prolonged hypoxia and/or nutrient withdrawal, which mimics posttransplantation environment of transplanted islets in the liver. We studied the influence of 24 h of hypoxia (1% O2) in intact isolated human and rat islets as well as the effect of combined oxygen/nutrient deprivation in a mouse insulinoma cell line (MIN6). In intact human islets, 24 h of hypoxia led to central necrosis combined with apoptotic features such as nuclear pyknosis and DNA fragmentation. In the course of hypoxic treatment, ultrastructural analysis demonstrated a gradual transition from an apoptotic to a necrotic morphology particularly pronounced in central areas of large islets. In MIN6 cells, on the other hand, hypoxia led to a twofold (p < 0.01) increase in caspase-3 activity, an indicator of apoptosis, but not to necrosis, as determined by release of lactate dehydrogenase (LDH). Only in combination with nutrient/serum deprivation was a marked increase in LDH release observed (sixfold vs. control, p < 0.01). We therefore conclude that, similar to MIN6 cells, central necrosis in isolated hypoxic islets is the result of the combined effects of hypoxia and nutrient/serum deprivation, most likely due to limited diffusion. Provided that transplanted islets undergo a similar fate as shown in our in vitro study, future emphasis will require the development of strategies that protect the islet graft from early cell death and accelerate the revascularization process.
Asunto(s)
Isquemia/fisiopatología , Islotes Pancreáticos/patología , Adenosina Trifosfato/metabolismo , Adulto , Animales , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , Hipoxia de la Célula/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Humanos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/ultraestructura , Trasplante de Islotes Pancreáticos , Masculino , Ratones , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Necrosis , Ratas , Ratas Sprague-DawleyRESUMEN
The bacteriolytic activity of CTL is mediated by granulysin, which has been reported to kill intracellular Mycobacterium tuberculosis in dendritic cells (DC) with high efficiency. Despite that crucial effector function, the killing mechanism and uptake of granulysin into target cells have not been well investigated. To this end we analyzed granulysin binding, uptake, and the subsequent lysis of intracellular Listeria innocua in human DC. Recombinant granulysin was found to be actively taken up by DC into early endosomal Ag 1-labeled endosomes, as detected by immunofluorescence. Further transfer to L. innocua-containing phagosomes was indicated by colocalization of bacterial DNA with granulysin. After uptake of granulysin by DC, lysis of L. innocua was found in a dose-dependent manner. Uptake as well as lysis of Listeria were inhibited after blocking endocytosis by lowering the temperature and by cholesterol depletion of DC. Colocalization of granulysin with cholera toxin during uptake showed binding to and internalization via lipid rafts. In contrast to cholera toxin, which was targeted to the perinuclear compartment, granulysin was found exclusively in endosomal-phagosomal vesicles. Lipid raft microdomains, enriched in the immunological synapse, may thus enhance uptake and transfer of granulysin into bacterial infected host cells.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/metabolismo , Bacteriólisis , Células Dendríticas/metabolismo , Listeria/fisiología , Microdominios de Membrana/metabolismo , Antígenos de Diferenciación de Linfocitos T/farmacología , Transporte Biológico , Células Cultivadas , Células Dendríticas/microbiología , Relación Dosis-Respuesta a Droga , Endosomas/metabolismo , Humanos , Microdominios de Membrana/inmunología , Microscopía Fluorescente , Fagosomas/metabolismoRESUMEN
BACKGROUND: The intracellular bacterium Chlamydia pneumoniae is suspected to play a role in formation and progression of atherosclerosis. Many studies investigated cell death initiation versus inhibition by Chlamydia pneumoniae in established cell lines but nothing is known in primary human aortic smooth muscle cells, a cell type among others known to be involved in the formation of the atherosclerotic plaque. Type of cell death was analyzed by various methods in primary aortic smooth muscle cells after infection with Chlamydia pneumoniae to investigate a possible pathogenic link in atherosclerosis. RESULTS: Chlamydiae were found to be localized up to 72 h post infection in aortic smooth muscle cells either as single bacteria or inside of large inclusions. Quantification of host cell death by lactate dehydrogenase release assay revealed strictly dose and time dependent lysis for all tested isolates of Chlamydia pneumoniae. Phosphatidylserine exposure was detected by flow cytometry in Chlamydia pneumoniae infected cells. Ultrastructure of Chlamydia pneumoniae infected human aortic smooth muscle cells showed extensive membrane- and organelle damage, chromatin condensation but no nuclear fragmentation. DNA fragmentation as well as cell membrane permeability was analyzed by TUNEL and NHS-biotin staining and occurred exclusively in cells carrying Chlamydia pneumoniae spots but not in smooth muscle cells with inclusions. These morphological features of cell death were not accompanied by an activation of caspase-3 as revealed by analysis of enzyme activity but involved mitochondrial membrane depolarization as shown by TMRE uptake and release of cytochrome c from mitochondria. CONCLUSION: This study provides evidence that Chlamydia pneumoniae induce a spot like infection in human aortic smooth muscle cells, which results in a chimeric cell death with both apoptotic and necrotic characteristics. This aponecrotic cell death may assist chronic inflammation in atherosclerotic blood vessels.
Asunto(s)
Aorta/patología , Chlamydophila pneumoniae/fisiología , Músculo Liso Vascular/microbiología , Músculo Liso Vascular/patología , Aorta/microbiología , Caspasa 3 , Caspasas/metabolismo , Línea Celular Tumoral , Núcleo Celular , Cromatina , Humanos , NecrosisRESUMEN
Listeria monocytogenes is the causative agent of infections like sepsis and meningitis, especially in immunocompromised hosts. Human macrophages are able to phagocytose and digest L. monocytogenes but IL-4 prevents human macrophages from killing the bacteria, the mechanisms of which are unknown. In the present study, we examined various listeria species and strains including wild-type and deletion mutants in human macrophages pretreated with IL-4. To analyse the IL-4-mediated deactivation process, we combined quantitative infection assays with various morphologic methods. IL-4 facilitates survival and escape of the pathogenic L. monocytogenes wild-type strain 10403S from the macrophage phagosomes. In untreated macrophages, the isogenic listeriolysin deletion mutant strain DP-L2161 was killed and did not escape from the phagolysosomes. However, after macrophage deactivation with IL-4 DP-L2161 survived and escaped from the phagosomes. This was also the case, but to a lesser extent, even for the naturally avirulent L. innocua. As detected by confocal laser-scanning fluorescence microscopy and electron microscopy, IL-4 permitted the escape of all listeria species tested, including DP-L2161 and L. innocua from the phagosomal compartment of the macrophages. We conclude that escape from the phagosome and survival of the listeria species tested in IL-4-deactivated human macrophages is independent of the virulence factor listeriolysin.
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Toxinas Bacterianas , Proteínas de Choque Térmico , Proteínas Hemolisinas , Interleucina-4/farmacología , Listeria monocytogenes/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Fagosomas/inmunología , Antibacterianos/farmacología , Toxinas Bacterianas/genética , Supervivencia Celular , Células Cultivadas , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Macrófagos/efectos de los fármacos , Microscopía Electrónica de Transmisión , Fagosomas/microbiología , VirulenciaRESUMEN
AIM: Animal protection laws will lead to stricter and more selective criteria thus resulting in a decline of available animals. Yet to train cardiac surgical skills a totally artificial training model was developed. DESCRIPTION OF THE TRAINING MODEL: The model is based on differently hardened polyurethane. Cover is a 1:1 replica of the human thoracic wall. Disposable coronaries are integrated in the heart-model. Vessels and part of the ascending aorta can be rinsed. By means of a newly designed air-pump stroke volume, heart-rate and rhythm can be adjusted. EXPERIENCES: Set-up of the model is easy and quick. Accustomed instruments can be used. Handling of artificial tissue is nature-like. Degree of difficulty is dependent on stroke volume, heart rate, arrhythmia, vessel-size and vessel-quality. CONCLUSION: The phantom helps to achieve confidence in coronary revascularisation. It facilitates an accompanying training for the less-trained as well as the skilled surgeon. The nature-like characteristics will help to reduce animal experiments in future.
Asunto(s)
Procedimientos Quirúrgicos Cardíacos/educación , Modelos Anatómicos , Revascularización Miocárdica/educación , Alternativas a las Pruebas en Animales , Competencia Clínica , Enfermedad Coronaria/cirugía , Vasos Coronarios/cirugía , Educación Médica Continua/métodos , HumanosRESUMEN
A new method for simultaneous mapping of cell topography and ion fluxes was developed. A highly sensitive ion sensor system was generated by coating atomic force microscopy tips with a PVC layer containing valinomycin, an ionophore for potassium. The activity of specific ions was traced on artificial ion-releasing PVC substrates. A boundary potential was generated owing to the selective exchange of a specific ion between coated tip and ion-releasing substrate. The boundary potential was detectable as a force induced by ion-selective electrostatic interactions. The selectivity coefficient of valinomycin for potassium against sodium (K(K,Na)f) was -2.5 +/- 0.5. Potassium efflux was measured on living MDCK-F1 cells expressing BK(Ca) channels. We could demonstrate localized areas of high potassium concentrations at the cell surface. The potassium efflux could be reversibly inhibited by thapsigargin, which is known to inhibit the efflux of potassium from BK(Ca) channels by suppression of calcium ATPase.
Asunto(s)
Técnicas Biosensibles/métodos , Células/química , Microscopía de Fuerza Atómica , Potasio/metabolismo , Línea Celular Transformada , Células/metabolismo , Humanos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Iones/metabolismo , Valinomicina/farmacologíaRESUMEN
Because of organ shortages in clinical allotransplantation, the potential of pig-to-human xenotransplantation is currently being explored showing a possible critical role for natural killer (NK) cells in the immune response against xenografts. Therefore, we analyzed the cytotoxic pathways utilized by human natural killer cells (hNK) against porcine endothelial cells (pEC). Transmission electron microscopy of pEC cocultured with hNK cells showed both apoptotic and necrotic cell death, whereas soluble factors such as Fas ligand or TNFalpha did not induce apoptosis in pEC. NK lysis of pEC was abrogated by concanamycin A and ammonium chloride, reagents inhibiting the perforin/granzyme B (grB) pathway, but only partially blocked by caspase inhibition with z-VAD-fmk. Overexpression of bcl-2 protected pEC against apoptosis induced by staurosporine or actinomycin D, but failed to prevent hNK cell-mediated lysis. In conclusion, pEC are lysed in vitro by hNK cells via the perforin/grB pathway and are not protected from NK lysis by overexpression of bcl-2.
Asunto(s)
Antígenos Heterófilos/inmunología , Endotelio Vascular/inmunología , Genes bcl-2 , Rechazo de Injerto/inmunología , Células Asesinas Naturales/inmunología , Macrólidos , Glicoproteínas de Membrana/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Serina Endopeptidasas/fisiología , Porcinos/inmunología , Clorometilcetonas de Aminoácidos/farmacología , Cloruro de Amonio/farmacología , Animales , Antibacterianos/farmacología , Apoptosis , Calcio/farmacología , Células Cultivadas/inmunología , Técnicas de Cocultivo , Cisteína Endopeptidasas/fisiología , Citotoxicidad Inmunológica , Dactinomicina/farmacología , Endotelio Vascular/ultraestructura , Inhibidores Enzimáticos/farmacología , Exocitosis/efectos de los fármacos , Proteína Ligando Fas , Granzimas , Humanos , Células Asesinas Naturales/ultraestructura , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/farmacología , Microscopía Electrónica , Necrosis , Perforina , Proteínas Citotóxicas Formadoras de Poros , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Recombinantes/farmacología , Estaurosporina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina/farmacologíaRESUMEN
Cytotoxic T lymphocytes mediate lysis of target cells by various mechanisms, including exocytosis of lytic proteins (perforin, granzymes) and receptor-ligand binding of Fas/APO molecules. Death of target cells is characterized by either necrosis or apoptosis, depending on the killing mechanism used and on the metabolism of the target cell itself.