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BACKGROUND AND OBJECTIVES: Blast-induced lung injury is associated with inflammatory, which are characterised by disruption of the alveolar-capillary barrier, haemorrhage, pulmonary infiltrateration causing oedema formation, pro-inflammatory cytokine and chemokine release, and anti-inflammatory counter-regulation. The objective of the current study was to define sequence of such alterations in with establishing blast-induced lung injury in rats using an advanced blast generator. METHODS: Rats underwent a standardized blast wave trauma and were euthanised at defined time points. Non-traumatised animals served as sham controls. Obtained samples from bronchoalveolar lavage fluid (BALF) at each time-point were assessed for histology, leukocyte infiltration and cytokine/chemokine profile. RESULTS: After blast lung injury, significant haemorrhage and neutrophil infiltration were observed. Similarly, protein accumulation, lactate dehydrogenase activity (LDH), alveolar eicosanoid release, matrix metalloproteinase (MMP)-2 and -9, pro-Inflammatory cytokines, including tumour necrosis factor (TNF) and interleukin (IL) -6 raised up. While declining in the level of anti-inflammatory cytokine IL-10 occurred. Ultimately, pulmonary oedema developed that increased to its maximum level within the first 1.5 h, then recovered within 24 h. CONCLUSION: Using a stablished model, can facilitate the study of inflammatory response to blast lung injury. Following the blast injury, alteration in cytokine/chemokine profile and activity of cells in the alveolar space occurs, which eventuates in alveolar epithelial barrier dysfunction and oedema formation. Most of these parameters exhibit time-dependent return to their basal status that is an indication to resilience of lungs to blast-induced lung injury.
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Lesión Pulmonar , Edema Pulmonar , Ratas , Animales , Lesión Pulmonar/etiología , Citocinas , Pulmón/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6 , EdemaRESUMEN
Objective: Current treatments for blast-induced lung injury are limited to supportive procedures including mechanical ventilation. The study aimed to investigate the role of post-trauma-induced oedema generation in the function of time and trauma intensity and the probable role of beta 2-adrenergic receptors (ß2-ARs) agonists on pulmonary oedema. The study is conducted using an ex vivo model after an experimental in vivo blast-induced thorax trauma in rats. Methods: Rats were randomised and divided into two groups, blast and sham. The blast group were anaesthetised and exposed to the blast wave (3.16 ± 0.43 bar) at a distance of 3.5 cm from the thorax level. The rats were sacrificed 10 min after the blast, the lungs explanted and treated with terbutaline, formoterol, propranolol or amiloride to assess the involvement of sodium transport. Other groups of rats were exposed to distances of 5 and 7 cm from the thorax to reduce the intensity of the injury. Further, one group of rats was studied after 180 min and one after 360 min after a 3.5 cm blast injury. Sham controls were exposed to identical procedures except for receiving blast overpressure. Results: Lung injury and oedema generation depended on time after injury and injury intensity. Perfusion with amiloride resulted in a further increase in oedema formation as indicated by weight gain (p < 0.001), diminished tidal volume (Tv) (p < 0.001), and increased airway resistance (p < 0.001). Formoterol caused a significant increase in the Tv (p < 0.001) and a significant decrease in the airway resistance (p < 0.01), while the lung weight was not influenced. Trauma-related oedema was significantly reduced by terbutaline in terms of lung weight gain (p < 0.01), Tv (p < 0.001), and airway resistance (p < 0.01) compared to control blast-injured lungs. Terbutaline-induced effects were completely blocked by the ß-receptor antagonist propranolol (p < 0.05). Similarly, amiloride, which was added to terbutaline perfusion, reversed terbutaline-induced weight gain reduction (p < 0.05). Conclusions: ß2-adrenoceptor stimulation had a beneficial impact by amiloride-dependent sodium and therefore, fluid transport mechanisms on the short-term ex vivo oedema generation in a trauma-induced in vivo lung injury of rats.
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RATIONALE: Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. OBJECTIVES: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). METHODS: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. MEASUREMENTS AND MAIN RESULTS: TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. CONCLUSIONS: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.
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Agonistas del Canal de Sodio Epitelial/metabolismo , Canales Epiteliales de Sodio/metabolismo , Péptidos Cíclicos/metabolismo , Alveolos Pulmonares/metabolismo , Edema Pulmonar/metabolismo , Estreptolisinas , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proteínas Bacterianas , Agonistas del Canal de Sodio Epitelial/química , Canales Epiteliales de Sodio/química , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Péptidos Cíclicos/química , Alveolos Pulmonares/microbiología , Edema Pulmonar/microbiología , Factor de Necrosis Tumoral alfa/químicaRESUMEN
BACKGROUND: Tumor necrosis factor alpha (TNF) is able to kill cancer cells via receptor-mediated cell death requiring adenosine triphosphate (ATP). Clinical usage of TNF so far is largely limited by its profound hepatotoxicity. Recently, it was found in the murine system that specific protection of hepatocytes against TNF's detrimental effects can be achieved by fructose-mediated ATP depletion therein. Before employing this quite attractive selection principle in a first clinical trial, we here comprehensively investigated the interdependence between ATP depletion and TNF hepatotoxicity in both in vitro and ex vivo experiments based on usage of primary patient tissue materials. METHODS: Primary human hepatocytes, and both non-tumorous and tumorous patient-derived primary liver tissue slices were used to elucidate fructose-induced ATP depletion and TNF-induced cytotoxicity. RESULTS: PHH as well as tissue slices prepared from non-malignant human liver specimen undergoing a fructose-mediated ATP depletion were both demonstrated to be protected against TNF-induced cell death. In contrast, due to tumor-specific overexpression of hexokinase II, which imposes a profound bypass on hepatocytic-specific fructose catabolism, this was not the case for human tumorous liver tissues. CONCLUSION: Normal human liver tissues can be protected transiently against TNF-induced cell death by systemic pretreatment with fructose used in non-toxic/physiologic concentrations. Selective TNF-targeting of primary and secondary tumors of the liver by transient and specific depletion of hepatocytic ATP opens up a new clinical avenue for the TNF-based treatment of liver cancers.
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Adenosina Trifosfato/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Fructosa/metabolismo , Fructosa/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hexoquinasa/metabolismo , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
Fructose-induced hepatic ATP depletion prevents TNF-induced apoptosis, whereas it contrarily enhances CD95-induced hepatocyte apoptosis in vitro and in vivo. By contrast, transformed liver cells are not protected against TNF due to metabolic alterations, allowing selective tumor targeting. We analyzed the molecular mechanisms by which fructose modulates cytokine-induced apoptosis. A release of adenosine after fructose-induced ATP depletion, followed by a cAMP response, was demonstrated. Likewise, cAMP and adenosine mimicked per se the modulation by fructose of CD95- and TNF-induced apoptosis. The effects of fructose on cytokine-induced apoptosis were sensitive to inhibition of protein kinase A. Fructose prevented the pro-apoptotic, sustained phase of TNF-induced JNK signaling and thereby blocked bid-mediated activation of the intrinsic mitochondrial apoptosis pathway in a PKA-dependent manner. We explain the dichotomal effects of fructose on CD95- and TNF-induced cell death by the selective requirement of JNK signaling for the latter. These findings provide a mechanistic rationale for the protection of hepatocytes from TNF-induced cell death by pharmacological doses of fructose.
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Apoptosis/efectos de los fármacos , Fructosa/farmacología , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Edulcorantes/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hepatocitos , Ratones , Receptor fas/metabolismoRESUMEN
We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.
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Proteínas Bacterianas/química , Chlamydophila pneumoniae/metabolismo , Fragmentos de Péptidos/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Antígenos Bacterianos/química , Biomarcadores/química , Chlamydophila pneumoniae/química , Inmunoensayo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Histone deacetylases (HD) represent a novel target in cancer treatment, particularly for scattered small tumours such as the hepatocellular carcinoma (HCC). However, only few studies address the toxicological impact of HD Inhibitors (HDIs) on malignantly transformed cells versus primary hepatocytes. We examined whether and how different classes of HDIs sensitise the human HCC cell line HepG2, primary healthy murine and human liver cells towards the death receptor agonists TNFα and CD95L. Apicidin, M344 (N-hydroxy-7-(-4-dimethylaminobenzol)aminoheptanamide), CBHA (m-carboxycinnamic acid bis-hydroxamide) and VPA (valproic acid) sensitised liver cell cultures towards CD95-triggered apoptosis with the following potency: apicidin > M344 ≈ CBHA â« VPA. Apicidin sensitised towards CD95 also in the intact organ, i.e. in the isolated perfused mouse liver. No significant sensitisation towards TNFα was found in vitro. Western blot analysis showed that all HDIs studied downregulated the anti-apoptotic protein cFLIP, but only VPA additionally affected the expression level of XIAP. Furthermore, in models of the intrinsic apoptosis pathway, i.e. in HepG2 cells treated with Melphalan and in primary hepatocytes irradiated with UV light, only VPA exhibited significant sensitisation. These findings extend the biochemical, pharmacological and toxicological basis for HDI therapy and provide a caveat for clinical use in patients with an accompanying critical inflammatory state in which the CD95 system might be pre-activated.
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Antineoplásicos/inmunología , Antineoplásicos/toxicidad , Hepatocitos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/inmunología , Inhibidores de Histona Desacetilasas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Proteína Ligando Fas/inmunología , Proteína Ligando Fas/metabolismo , Células Hep G2 , Hepatocitos/enzimología , Hepatocitos/inmunología , Histona Desacetilasas/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Perfusión , Transfección , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND & AIMS: TNF was the first cytokine employed for cancer therapy, but its use was limited due to its insufficient selectivity towards malignant cells. Fructose induces transient hepatic ATP depletion in humans and rodents due to the liver-specific fructose metabolism via fructokinase, while other cells e.g. Muscle cells metabolize fructose via hexokinase. Under ATP depleted conditions hepatocytes are protected against TNF-induced apoptosis. Our aim was to identify metabolic differences between normal and malignant liver cells that can be exploited for selective immunotherapy. METHODS: We analyzed the expression and activities of enzymes involved in fructose metabolism in primary hepatocytes and hepatoma cell lines. Furthermore, we studied the influence of hexokinase II (HKII) on fructose-mediated ATP depletion and cytoprotection in murine hepatocytes. RESULTS: Primary mouse, rat and human hepatocytes depleted of ATP by fructose were fully protected against TNF-induced cytotoxicity. By contrast, hepatic tumor cell lines showed increased HKII expression, lack of fructose-mediated ATP depletion and, therefore, remained susceptible to TNF/ActD-induced apoptosis. Inhibition of hexokinases restored fructose-induced ATP depletion in hepg2 cells. Finally, hypoxia-inducible factor1 (HIF1)-mediated up-regulation of HKII prevented fructose-induced ATP depletion and overexpression of HKII inhibited fructose-mediated cytoprotection against TNF-induced apoptosis in primary murine hepatocytes. CONCLUSION: Increased expression of HKII in malignant cells of hepatic origin shifts the fructose metabolism from liver- to muscle-type, thereby preventing ATP depletion and subsequent cytoprotection of the target cells. Therefore, healthy liver cells are transiently protected from TNF-mediated cell death by fructose-induced ATP depletion, while malignant cells can be selectively eliminated through TNF-induced apoptosis.
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Adenosina Trifosfato/metabolismo , Fructosa/farmacología , Hepatocitos/efectos de los fármacos , Neoplasias Hepáticas/patología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Citoprotección , Fructoquinasas/fisiología , Fructosa-Bifosfato Aldolasa/fisiología , Hexoquinasa/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Ratones , RatasRESUMEN
Listeriosis can lead to potentially lethal pulmonary complications in newborns and immune compromised patients, characterized by extensive permeability edema. Listeriolysin (LLO), the main virulence factor of Listeria monocytogenes, induces a dose-dependent hyperpermeability in monolayers of human lung microvascular endothelial cells in vitro. The permeability increasing activity of LLO, which is accompanied by an increased reactive oxygen species (ROS) generation, RhoA activation and myosin light chain (MLC) phosphorylation, can be completely inhibited by the protein kinase C (PKC) alpha/beta inhibitor GO6976, indicating a crucial role for PKC in the induction of barrier dysfunction. The TNF-derived TIP peptide, which mimics the lectin-like domain of the cytokine, blunts LLO-induced hyperpermeability in vitro, upon inhibiting LLO-induced protein kinase C-alpha activation, ROS generation and MLC phosphorylation and upon restoring the RhoA/Rac 1 balance. These results indicate that the lectin-like domain of TNF has a potential therapeutic value in protecting from LLO-induced pulmonary endothelial hyperpermeability.
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Toxinas Bacterianas/toxicidad , Endotelio Vascular/metabolismo , Proteínas de Choque Térmico/toxicidad , Proteínas Hemolisinas/toxicidad , Listeria monocytogenes/patogenicidad , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/microbiología , Humanos , Pulmón/citología , Pulmón/metabolismo , Pulmón/microbiología , Cadenas Ligeras de Miosina/metabolismo , Péptidos/farmacología , Permeabilidad , Fosforilación , Proteína Quinasa C-alfa/antagonistas & inhibidores , Arteria Pulmonar/metabolismo , Arteria Pulmonar/microbiología , Especies Reactivas de Oxígeno/metabolismo , Ovinos , Factor de Necrosis Tumoral alfa/química , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
OBJECTIVE: To test the hypothesis that the lectin-like domain of tumor necrosis factor, mimicked by the TIP peptide, can improve lung function after unilateral orthotopic lung isotransplantation. Because of a lack of a specific treatment for ischemia reperfusion-mediated lung injury, accompanied by a disrupted barrier integrity and a dysfunctional alveolar liquid clearance, alternative therapies restoring these parameters after lung transplantation are required. DESIGN: Prospective, randomized laboratory investigation. SETTING: University-affiliated laboratory. SUBJECTS: Adult female rats. INTERVENTIONS: Tuberoinfundibular peptide, mimicking the lectin-like domain of tumor necrosis factor, mutant TIP peptide, N,N'-diacetylchitobiose/TIP peptide, and amiloride/TIP peptide were instilled intratracheally in the left lung immediately before the isotransplantation was performed. An additional group received an intravenous TIP peptide treatment, 1.5 mins before transplantation. Studies using isolated rat type II alveolar epithelial cell monolayers and ovine pulmonary endothelial cells were also performed. MEASUREMENTS AND MAIN RESULTS: Intratracheal pretreatment of the transplantable left lung with the TIP peptide, but not with an inactive mutant TIP peptide, resulted in significantly improved oxygenation 24 hrs after transplantation. This treatment led to a significantly reduced neutrophil content in the lavage fluid. Both the effects on oxygenation and neutrophil infiltration were inhibited by the epithelial sodium channel blocker amiloride. The TIP peptide blunted reactive oxygen species production in pulmonary artery endothelial cells under hypoxia and reoxygenation and reduced reactive oxygen species content in the transplanted rat lungs in vivo. Ussing chamber experiments using monolayers of primary type II rat pneumocytes indicated that the primary site of action of the peptide was on the apical side of these cells. CONCLUSIONS: These data demonstrate that the TIP peptide significantly improves lung function after lung transplantation in the rat, in part, by reducing neutrophil content and reactive oxygen species generation. These studies suggest that the TIP peptide is a potential therapeutic agent against the ischemia reperfusion injury associated with lung transplantation.
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Trasplante de Pulmón/fisiología , Pulmón/irrigación sanguínea , Neuropéptidos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/fisiopatología , Pruebas de Función Respiratoria , Factor de Necrosis Tumoral alfa/farmacología , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/fisiología , Amilorida/farmacología , Animales , Disacáridos/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Oxígeno/fisiología , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/fisiopatología , Ratas , Ovinos , Bloqueadores de los Canales de Sodio/farmacología , Superóxidos/metabolismoRESUMEN
Beta2-agonists have been shown to increase alveolar fluid reabsorption, and at least part of their effect depends on active sodium transport from the alveolus into the epithelial cell by the amiloride-sensitive epithelial sodium channel (ENaC). Few data exist on their effect in the injured lung. The authors therefore investigated the effect of intrabronchially administered terbutaline pretransplantation by measuring outcome 1 day after experimental donor lung transplantation with severe injury due to prolonged ischemia. Orthotopic single left-sided lung allotransplantation was performed in female rats (Wistar to Wistar) after a total ischemic time of 20 hours. Graft PaO2/FiO2 in 6 recipients treated with 10(-4) M terbutaline in 500 microL NaCl 0.9% was superior 24 hours after transplantation, with a PaO2 of 329 (111 [SD]) mm Hg versus 5 vehicle controls with 44 (15) mm Hg (P = .002). The beneficial effect of 10(-4) M terbutaline was abrogated by 10(-4) M of the sodium channel blocker amiloride to 71 (34) mm Hg in 3 recipients (P = .028 versus terbutaline 10(-4) M). Ten recipients receiving 10(-5) M terbutaline in 500 microL NaCl 0.9% showed inconsistent improvements of gas exchange, with a PaO2 of 158 (+/- 153) mm Hg (P = .058). Terbutaline at a high dose significantly improved the transplanted rat lung function at 24 hours after transplantation. Part of it may be via activating epithelial sodium transport, thus suggesting an important role of alveolar fluid transport in such a model of acute lung injury.
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Trasplante de Pulmón/efectos adversos , Daño por Reperfusión/tratamiento farmacológico , Terbutalina/farmacología , Agonistas Adrenérgicos beta/farmacología , Agonistas Adrenérgicos beta/uso terapéutico , Animales , Transporte Biológico , Femenino , Oxígeno/metabolismo , Alveolos Pulmonares/metabolismo , Ratas , Ratas Wistar , Sodio/metabolismo , Terbutalina/uso terapéutico , Resultado del TratamientoRESUMEN
The heterocyclic organic compound ebselen (2-phenyl-1,2-benizsoselenazol-3(2H)-one) is a glutathione peroxidase mimick with protective properties against oxidative injury. Ebselen also has anti-inflammatory activity, including attenuation of tumor necrosis factor release and increase of interleukin-10, as shown in vivo, in inflammatory and ischemia-reperfusion injuries, including those of the lung. This study was designed to assess its effect on severe ischemia-reperfusion injury in a model of left-sided rat lung isotransplantation. Orthotopic single left-sided lung allotransplantation (Wistar to Wistar) was performed in female rats after a total ischemic time of 18 h. In nine recipients given 500 mg/kg oral ebselen 1 h before transplantation, graft PaO(2)/FiO(2) was improved 24 h after transplantation, as evidenced with a mean (standard deviation) PaO(2) of 139 (61) mmHg vs. eight controls with 65 (33) mmHg (p = 0.009). Bronchoalveolar PMN count was reduced to approximately 50% in the ebselen group compared with controls, whereas no difference in the tumor necrosis factor content was found. We conclude that the improvement of lung function in ebselen-treated transplanted rats is mainly the result of the anti-inflammatory activity of the drug during reperfusion.
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Lesión Pulmonar Aguda/prevención & control , Antiinflamatorios no Esteroideos/farmacología , Azoles/farmacología , Trasplante de Pulmón/efectos adversos , Pulmón/efectos de los fármacos , Compuestos de Organoselenio/farmacología , Daño por Reperfusión/prevención & control , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/fisiopatología , Administración Oral , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Azoles/administración & dosificación , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Femenino , Interleucina-10/metabolismo , Isoindoles , Pulmón/inmunología , Pulmón/fisiopatología , Infiltración Neutrófila/efectos de los fármacos , Compuestos de Organoselenio/administración & dosificación , Intercambio Gaseoso Pulmonar/efectos de los fármacos , Ratas , Ratas Wistar , Daño por Reperfusión/etiología , Daño por Reperfusión/inmunología , Daño por Reperfusión/fisiopatología , Índice de Severidad de la Enfermedad , Trasplante Homólogo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
5-Azacytidine (5-aza-CR) is a DNA-hypomethylating antineoplastic agent used because of its inhibitory activity on DNA methyltransferases. Today, it is approved as an epigenetically active drug therapy for treatment of myelodysplastic disorders, with a contraindication as to pre-existing liver diseases. Because the mechanism of its hepatotoxicity is still unknown, we investigated the pharmacodynamic properties of 5-aza-CR with regard to death receptor/ligand-induced apoptosis and the mode of execution of cell death. In a time- and concentration-dependent manner, primary murine, human hepatocytes and HepG2 cells exposed to 5-aza-CR became highly sensitive toward cell death induced by CD95L, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, or TNF. Cell death was characterized as apoptotic by membrane blebbing, chromatin condensation, and exposure of phosphatidylserine on the outer membrane. Neither 5-aza-2'-deoxycytidine nor the common DNA methyltransferase inhibitors S-(5'-adenosyl)-L-homocysteine or RG 108 showed any significant effects under these conditions. Despite the complete protection of HepG2 by high concentrations of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-fmk), effector caspase-3/7 activity was completely abolished at approximately a 20-fold lower concentration of z-VAD-fmk. Under these conditions, the serine protease inhibitors N,alpha-tosyl-L-phenylalanine chloromethyl ketone, N,p-tosyl-L-lysine chloromethyl ketone, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, respectively, conferred protection against death receptor ligands. We conclude that this caspase-independent apoptosis is executed by a yet-unidentified serine protease.
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Apoptosis/fisiología , Azacitidina/farmacología , Hepatocitos/fisiología , Hígado/citología , Hígado/fisiología , Receptores de Muerte Celular/agonistas , Receptores de Muerte Celular/fisiología , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Técnicas de Cultivo de Célula , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Hígado/efectos de los fármacos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/cirugía , Ratones , Microscopía FluorescenteRESUMEN
The remarkable ability of TNF, especially in combination with Interferon-gamma or melphalan, to inhibit the growth of malignant tumor cells is so far unmatched. Unfortunately, its high systemic toxicity and hepatotoxicity prevent its systemic use in cancer patients. An elegant manner to circumvent this problem is the isolated limb and liver perfusion for the treatment of melanoma, soft tissue sarcoma and liver tumors, respectively, although the latter method can lead to a reversible hepatotoxicity. In order to allow also the treatment of other cancers with TNF, new strategies have to be developed that aim at sensitizing tumor cells to TNF and at reducing its systemic and liver toxicity, without losing its antitumor efficiency. Moreover, the lectin-like domain of TNF, which is spatially distinct from the receptor binding sites, could be useful in reducing cancer treatment-related pulmonary edema formation. This review will discuss some recent developments in these areas, which can lead to a renewed interest in TNF for the systemic treatment of cancer.
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Infecciones/fisiopatología , Neoplasias/fisiopatología , Factor de Necrosis Tumoral alfa/fisiología , Adenosina Trifosfato/metabolismo , Antineoplásicos/uso terapéutico , Endotelio Vascular/fisiopatología , Humanos , Inflamación/fisiopatología , Leucocitos/fisiología , Hígado/metabolismo , Necrosis , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Factor de Necrosis Tumoral alfa/uso terapéutico , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
BACKGROUND: The dialysis patient is confronted with hundreds of litres of dialysis solution per week, which pass the natural protective barriers of the body and are brought into contact with the tissue directly in the case of peritoneal dialysis or indirectly in the case of renal dialysis (hemodialysis). The components can be tested for living specimens or dead pyrogenic (fever-inducing) contaminations. The former is usually detected by cultivation and the latter by the endotoxin-specific Limulus Amoebocyte Lysate Assay (LAL). However, the LAL assay does not reflect the response of the human immune system to the wide variety of possible pyrogenic contaminations in dialysis fluids. Furthermore, the test is limited in its sensitivity to detect extremely low concentrations of pyrogens, which in their sum result in chronic pathologies in dialysis patients. The In vitro Pyrogen Test (IPT) employs human whole blood to detect the spectrum of pyrogens to which humans respond by measuring the release of the endogenous fever mediator interleukin-1beta. Spike recovery checks exclude interference. The test has been validated in an international study for pyrogen detection in injectable solutions. METHODS: In this study we adapted the IPT to the testing of dialysis solutions. RESULTS: Preincubation of 50 ml spiked samples with albumin-coated microspheres enhanced the sensitivity of the assay to detect contaminations down to 0.1 pg/ml LPS or 0.001 EU/ml in water or saline and allowed pyrogen detection in dialysis concentrates or final working solutions. CONCLUSIONS: This method offers high sensitivity detection of human-relevant pyrogens in dialysis solutions and components.
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Soluciones para Diálisis/análisis , Interleucina-1beta/análisis , Pirógenos/análisis , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/aislamiento & purificación , Humanos , Lipopolisacáridos/química , Lipopolisacáridos/aislamiento & purificación , Microesferas , Pirógenos/aislamiento & purificación , Albúmina Sérica/química , Staphylococcus aureus/aislamiento & purificación , Ácidos Teicoicos/química , Ácidos Teicoicos/aislamiento & purificaciónRESUMEN
The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.
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Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Infecciones por Chlamydia/inmunología , Chlamydophila pneumoniae/inmunología , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/aislamiento & purificación , Western Blotting , Infecciones por Chlamydia/microbiología , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , ProteómicaRESUMEN
BACKGROUND: Bone marrow-derived progenitors for both epithelial and endothelial cells have been observed in the lung. Besides mature endothelial cells (EC) that compose the adult vasculature, endothelial progenitor cells (EPC) are supposed to be released from the bone marrow into the peripheral blood after stimulation by distinct inflammatory injuries. Homing of ex vivo generated bone marrow-derived EPC into the injured lung has not been investigated so far. We therefore tested the hypothesis whether homing of EPC in damaged lung tissue occurs after intravenous administration. METHODS: Ex vivo generated, characterized and cultivated rat bone marrow-derived EPC were investigated for proliferation and vasculogenic properties in vitro. EPC were tested for their homing in a left-sided rat lung transplant model mimicking a severe acute lung injury. EPC were transplanted into the host animal by peripheral administration into the femoral vein (10(6) cells). Rats were sacrificed 1, 4 or 9 days after lung transplantation and homing of EPC was evaluated by fluorescence microscopy. EPC were tested further for their involvement in vasculogenesis processes occurring in subcutaneously applied Matrigel in transplanted animals. RESULTS: We demonstrate the integration of intravenously injected EPC into the tissue of the transplanted left lung suffering from acute lung injury. EPC were localized in vessel walls as well as in destructed lung tissue. Virtually no cells were found in the right lung or in other organs. However, few EPC were found in subcutaneous Matrigel in transplanted rats. CONCLUSION: Transplanted EPC may play an important role in reestablishing the endothelial integrity in vessels after severe injury or at inflammatory sites and might further contribute to vascular repair or wound healing processes in severely damaged tissue. Therapeutic applications of EPC transplantation may ensue.
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Células de la Médula Ósea/patología , Trasplante de Médula Ósea/métodos , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/patología , Neovascularización Fisiológica , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/cirugía , Animales , Femenino , Infusiones Parenterales , Ratas , Ratas Sprague-DawleyRESUMEN
UNLABELLED: Caspases are thought to be essential in execution of death receptor-induced apoptosis. However, recent findings suggest the existence of alternative pathways independent of caspases. We provide further evidence for such signaling in hepatocytes. RESULTS: Death receptor-induced activation of caspases and apoptosis in primary murine hepatocytes was completely blocked in presence of 1.5 microM N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk). Whereas the same concentration of the inhibitor was sufficient to block TNF receptor 1-, CD95- or TRAIL receptor 1/-2-induced activation of caspases in primary human hepatocytes or HepG2 cells, complete prevention apoptotic cell death needed almost 100 microM zVAD-fmk. Under caspase-inhibitory but non-protective conditions, i.e. at 1.5 microM zVAD-fmk, various serine protease inhibitors prevented apoptosis-like cell death. Neither sole arrest of caspases nor inhibition of serine proteases alone protected human hepatocytes. CONCLUSION: Human but not murine hepatocytes bear the potential to activate a permissive, serine protease inhibitor-sensitive alternative death signaling pathway under caspase-inhibitory conditions.
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Clorometilcetonas de Aminoácidos/administración & dosificación , Apoptosis/fisiología , Inhibidores de Caspasas , Caspasas/metabolismo , Hepatocitos/metabolismo , Receptores de Muerte Celular/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Humanos , Ratones , Complejos Multienzimáticos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Transducción de Señal/efectos de los fármacos , Especificidad de la EspecieRESUMEN
We demonstrated previously that depletion of hepatic ATP by endogenous metabolic shunting of phosphate after fructose treatment renders hepatocytes resistant to tumor necrosis factor (TNF)-induced apoptosis. We here address the question whether this principle extends to TNF receptor 1-mediated caspase-independent apoptotic and to necrotic liver injury. As in the apoptotic model of galactosamine/lipopolysaccharide (LPS)-induced liver damage, the necrotic hepatotoxicity initiated by sole high-dose LPS treatment was abrogated after depletion of hepatic ATP. Although systemic TNF and interferon-gamma levels were suppressed, animals still were protected when ATP depletion was initiated after the peak of proinflammatory cytokines upon LPS injection, showing that fructose-induced ATP depletion affects both cytokine release and action. In T cell-dependent necrotic hepatotoxicity elicited by concanavalin A or galactosamine + staphylococcal enterotoxin B, ATP depletion prevented liver injury as well, but here without modulating cytokine release. By attenuating caspase-8 activation, ATP depletion of hepatocytes in vitro impaired TNF receptor signaling by the death-inducing signaling complex, whereas receptor internalization and nuclear factor-kappaB activation upon TNF stimulation were unaffected. These findings demonstrate that sufficient target cell ATP levels are required for the execution of both apoptotic and necrotic TNF-receptor 1-mediated liver cell death.
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Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Hexosas/farmacología , Hígado/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Adenosina/farmacología , Alanina Transaminasa/sangre , Animales , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dactinomicina/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Interferón gamma/sangre , Interleucina-4/sangre , Lipopolisacáridos/farmacología , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , FN-kappa B/metabolismo , Necrosis/prevención & control , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Medical devices manufactured for implantation into humans must be free of any contamination with viable bacteria. However, remnants of dead bacteria and bacterial components alone may induce an inflammatory immune response. Pyrogen tests for such inflammatory contaminations are generally performed either by determining the content of lipopolysaccharide in rinsing solutions of batch samples by limulus amoebocyte lysate assay, by injecting the rinsing solutions into rabbits or by implanting batch samples into rabbits and measuring change of body temperature. In this study, we show that the in vitro pyrogen test (IPT), which measures the release of the inflammatory cytokine IL-1beta in fresh or cryopreserved human whole blood, can be used to assess the pyrogenic contamination of implantable medical devices. This test was used to check neurosurgical implants, namely aneurysm clips, as a proof of principle. Owing to the direct contact of the test material with the blood cells, this test does not require rinsing procedures, which have variable efficacy. The use of human blood ensures the detection of all substances that are pyrogenic for humans and reflects their relative potency. The safety of the products as delivered could be confirmed. The effects of sterilization and depyrogenization procedures on intentional pyrogenic contaminations of samples could be followed. This new application of the already internationally validated method promises to replace further rabbit pyrogen tests. It generates extremely sensitive results with an extended range of detectable pyrogenic contaminants.