RESUMEN
Pseudomonas aeruginosa possesses an arsenal of both cell-associated (flagella, pili, alginate, etc.) and extracellular (exotoxin A, proteases, type III secretion effectors, etc.) virulence factors. Among them, secreted proteases that damage host tissues are considered to play an important role in the pathogenesis of P. aeruginosa infections. We previously reported that EprS, an autotransporter protease of P. aeruginosa, induces host inflammatory responses through protease-activated receptors. However, little is known about the role of EprS as a virulence factor of P. aeruginosa. In this study, to investigate whether EprS participates in the pathogenicity of P. aeruginosa, we characterized various pathogenic phenotypes of the wild-type PAO1 strain and its eprS-disrupted mutant. The growth assays demonstrated that the growth of the eprS mutant was somewhat lower than that of the wild-type strain in a minimal medium containing BSA as the sole carbon and nitrogen source. Thus, these results indicate that eprS would have a role in the growth of P. aeruginosa in the presence of limited nutrients, such as a medium containing proteinaceous materials as a sole nutrient source. Furthermore, disruption of eprS resulted in a decreased production of elastase, pigments, autoinducers and surfactants, and a reduction of swimming and swarming motilities. In addition, the eprS mutant exhibited a reduction in the ability to associate with A549 cells and an attenuation of virulence in leucopenic mice as compared with the wild-type strain. Collectively, these results suggest that EprS exerts pleiotropic effects on various pathogenic phenotypes of P. aeruginosa.
Asunto(s)
Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , Serina Proteasas/genética , Factores de Virulencia/genética , Animales , Adhesión Bacteriana/genética , Línea Celular , Humanos , Ratones , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Tensoactivos/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: Cryopreservation of microorganism cultures is an important technology for their use as biological and genetic resources; however, the procedure is complicated and depends on the species. MATERIALS AND METHODS: We used the two-step freezing method for the cryopreservation of the green alga Parachlorella kessleri. RESULTS AND CONCLUSION: The optimal cryoprotectant for cryopreservation was 5% dimethyl sulfoxide plus 5% ethylene glycol. This is different from the optimal cryoprotectant for the closely related species Chlorella vulgaris. Efficient cryopreservation of P. kessleri was achieved using methanol, similar to Chlamydomonas reinhardtii. A membrane-specific fluorescent dye, FM1-43, was applied to estimate plasma membrane integrity. We found that the plasma membrane integrity of P. kessleri cells after freeze-thawing was associated with survivability, suggesting that this is a useful index for the optimization of the first step of the two-step freezing method of cryopreservation.
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Chlorophyta/citología , Criopreservación , Colorantes Fluorescentes/análisis , Compuestos de Piridinio/análisis , Compuestos de Amonio Cuaternario/análisis , Membrana Celular/química , Chlorophyta/química , Crioprotectores/química , Dimetilsulfóxido/química , Glicol de Etileno/química , CongelaciónRESUMEN
BACKGROUND: Airway viral infections provoke exacerbations of asthma and chronic obstructive pulmonary disease. B7-H1 is a costimulatory molecule that is implicated in an escape mechanism of viruses from host immune systems. This escape may be associated with the persistence of viral infection and lead to exacerbation of underlying diseases. We have shown that an analog of viral double-stranded RNA, polyinosinic-polycytidylic acid (poly IC), upregulated the expression of B7-H1 on airway epithelial cells, an effect which was corticosteroid-resistant. We investigated the effects of corticosteroids plus long-acting ß(2)-agonists (LABAs; fluticasone/salmeterol or budesonide/formoterol) on the expression of B7-H1. METHODS: BEAS-2B cells and primary airway epithelial cells were stimulated with poly IC or respiratory syncytial virus. The expression of B7-H1 was assessed by flow cytometry. RESULTS: Poly IC upregulated the expression of B7-H1, which was suppressed by high-concentration corticosteroids but not by LABAs. The upregulation was suppressed by very low-concentration corticosteroids when used in combination with LABAs. Their combination also suppressed the virus-induced upregulation of B7-H1. Poly IC stimulation induced the nuclear translocation of nuclear factor ĸB (NF-ĸB). Inhibitors of NF-ĸB activation prevented the poly IC-induced upregulation of B7-H1. Low-concentration corticosteroids in combination with LABAs enhanced the de novo induction of IĸBα, the endogenous inhibitor of NF-ĸB activation. CONCLUSIONS: Fluticasone/salmeterol or budesonide/formoterol attenuate the virus-associated upregulation of B7-H1 on airway epithelial cells via suppression of NF-ĸB activation.
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Corticoesteroides/farmacología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Antígeno B7-H1/metabolismo , Poli I-C/farmacología , Mucosa Respiratoria/metabolismo , Albuterol/análogos & derivados , Albuterol/farmacología , Androstadienos/farmacología , Budesonida/farmacología , Línea Celular , Combinación de Medicamentos , Etanolaminas/farmacología , Combinación Fluticasona-Salmeterol , Fumarato de Formoterol , Humanos , Proteínas I-kappa B/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/metabolismo , Virus Sincitiales Respiratorios/patogenicidad , Regulación hacia ArribaRESUMEN
We report the discovery of a family of ternary platinum phosphides APt3P (A = Ca, Sr, and La), which crystallize in an antiperovskite-based structure closely related to that of the heavy fermion superconductor CePt3Si. All three phosphides showed superconductivity at low temperatures and the highest critical temperature T(c) = 8.4 K was observed for SrPt3P. The analysis of specific heat C(T) for SrPt3P shows clear evidence for very strong coupling s-wave superconductivity with a large ratio between superconducting gap Δ0 and T(c), 2Δ0/k(B)T(c) â¼ 5, and the presence of low-energy phonons. The presence of multiple Fermi surface pockets was inferred from the nonlinear magnetic field dependence of Hall resistivity, which we argue might play a role in realizing the strong coupling of charge carriers with the low-lying phonons.
RESUMEN
BACKGROUND: Wild-type p53 is increased during cellular responses to various stresses. Mdm2, which is induced by p53, regulates p53 protein concentrations through the ubiquitin-proteasome pathway. AIM: To investigate whether the Mdm2 mediated ubiquitination of p53 is associated with epithelial cell apoptosis in idiopathic pulmonary fibrosis (IPF). METHODS: Immunohistochemistry and western blot analysis were carried out on lung samples obtained by lung biopsy from patients with IPF and non-specific interstitial pneumonia (NSIP). RESULTS: The expression of p53, phosphorylated p53, Mdm2, p21, and Bax was upregulated in epithelial cells from patients with IPF and NSIP compared with normal lung parenchyma. Except for p21, there was a significant increase in the expression of these factors in IPF compared with NSIP. In addition, the number of apoptotic cells and the number of p53 and Bax positive cells was increased compared with controls. p53 conjugated with Mdm2 was decreased in IPF compared with NSIP and controls. Ubiquitinated p53 was increased in both IPF and NSIP compared with controls. CONCLUSIONS: Signalling molecules associated with p53 mediated apoptosis may participate in epithelial cell apoptosis, and the attenuation of p53-Mdm2 conjugation and of p53 degradation may be involved in the epithelial cell apoptosis seen in IPF. Augmented epithelial apoptosis in IPF may lead to the poor prognosis compared with NSIP.
Asunto(s)
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Fibrosis Pulmonar/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Apoptosis , Western Blotting/métodos , Proteínas de Ciclo Celular/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Humanos , Pulmón/metabolismo , Enfermedades Pulmonares Intersticiales/metabolismo , Enfermedades Pulmonares Intersticiales/patología , Masculino , Persona de Mediana Edad , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Fibrosis Pulmonar/patología , Transducción de Señal , Ubiquitina/metabolismo , Regulación hacia Arriba , Proteína X Asociada a bcl-2RESUMEN
Evidence that apoptosis plays an important role in the pathophysiology of lung diseases has been accumulated. Apoptosis signaling is classically composed of two principle pathways. One is a direct pathway from death receptor ligation to caspase cascade activation and cell death. Death receptor ligation triggers recruitment of the precursor form of caspase-8 to a death-inducing complex, through the adaptor protein FADD, which leads to caspase-8 activation. The other pathway triggered by stimuli such as drugs, radiation, infectious agents and reactive oxygen species is initiated in mitochondria. After cytochrome c is released into the cytosol from the mitochondria, it binds to Apaf1 and ATP, which then activate caspase-9. Recently, endoplasmic reticulum has also been shown to be the organelle to execute apoptosis. Further understanding of molecular mechanisms of apoptosis and its regulation by novel drugs may lead to the development of effective strategies against lung diseases. We overview the signaling pathways of apoptosis and discuss the involvement of apoptosis in the pathophysiology of various lung diseases.
Asunto(s)
Apoptosis , Enfermedades Pulmonares/fisiopatología , Transducción de Señal , Animales , Asma/fisiopatología , Humanos , Modelos Biológicos , Enfisema Pulmonar/fisiopatología , Fibrosis Pulmonar/fisiopatologíaRESUMEN
AIMS: The perforin mediated pathway is the major pathway of cytotoxicity induced by activated T cells and natural killer cells, and may be involved in the development of pulmonary fibrosis. METHODS: Perforin and granzyme B expression were examined in idiopathic pulmonary fibrosis by means of immunohistochemistry, and perforin knockout mice were used to examine whether or not perforin mediated cytotoxicity participates in the pathophysiology of bleomycin induced pneumopathy. RESULTS: Perforin and granzyme B expression were upregulated in infiltrating lymphocytes in lung tissue from patients with idiopathic pulmonary fibrosis compared with normal lung parenchyma. Perforin and granzyme B expression were upregulated predominantly in infiltrating mononuclear cells after bleomycin instillation in wild-type mice. Although the development of bleomycin induced pneumopathy was not completely prevented, the pathological grade of inflammation and fibrosis, and the number of apoptotic cells in lung tissue, were significantly decreased in perforin knockout mice compared with wild-type mice. CONCLUSIONS: These results suggest that perforin mediated apoptosis may be associated with the pathophysiology of lung injury and fibrosis.
Asunto(s)
Apoptosis/fisiología , Enfermedades Pulmonares/fisiopatología , Glicoproteínas de Membrana/fisiología , Fibrosis Pulmonar/fisiopatología , Animales , Antimetabolitos Antineoplásicos , Apoptosis/inmunología , Bleomicina , Citotoxicidad Inmunológica/inmunología , Citotoxicidad Inmunológica/fisiología , Modelos Animales de Enfermedad , Granzimas , Inmunohistoquímica/métodos , Etiquetado Corte-Fin in Situ/métodos , Células Asesinas Naturales/inmunología , Enfermedades Pulmonares/inmunología , Glicoproteínas de Membrana/análisis , Ratones , Ratones Noqueados , Perforina , Proteínas Citotóxicas Formadoras de Poros , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/inmunología , ARN Mensajero/análisis , Serina Endopeptidasas/análisis , Linfocitos T Citotóxicos/inmunología , Regulación hacia Arriba/inmunología , Regulación hacia Arriba/fisiologíaRESUMEN
Drugs have been implicated in lung injury as a result of direct pharmacological action, persistence or metabolism in the tissue, or via the production of a reactive metabolite or metabolites. The result of this apparent drug-associated injury ranges from cellular dysfunction through to cell death (apoptosis) and alteration of repair mechanisms that are essential in replacing critical tissue elements and function. There is limited knowledge on how timing of drug administration or drug interactions may interfere with the repair mechanisms or modulate the expression of pulmonary toxicity. Chemotherapeutic drugs and novel agents, such as those targeting the epidermal growth factor receptor (EGFR), appear to affect both normal and neoplastic cells. However, unlike chemotherapy, where the actions are systemic and directly as a result of biotransformation or cell injury, it has been postulated that effects of EGFR-targeting agents are more likely to be focused on epithelia via a pharmacological effect. Furthermore, risk factors for the development of adverse pulmonary reactions, as well as biological markers indicating incipient toxicity, need to be prospectively identified. Proteomics, through the identification of >/=1000 proteins or peptides in blood samples, will hopefully identify candidates for this role.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Enfermedades Pulmonares Intersticiales/inducido químicamente , Enfermedades Pulmonares Intersticiales/fisiopatología , Neoplasias Pulmonares/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Apoptosis/efectos de los fármacos , Biomarcadores/análisis , Biotransformación , Terapia Combinada , Células Epiteliales , Receptores ErbB/antagonistas & inhibidores , Humanos , Proteómica , Traumatismos por Radiación , Factores de RiesgoRESUMEN
Pulmonary fibrosis is a common response to various injuries to the lung. The resolution of a fibroproliferative response after lung injury is key to survival. Although there are various initiating factors or causes, the terminal stages are characterized by proliferation and progressive accumulation of connective tissue replacing normal functional parenchyma. Conventional therapy consisting of glucocorticoids or immunosuppressive drugs is usually ineffective in preventing progression of fibrosis. Further understanding of the molecular mechanisms of endothelial and epithelial cell injury, inflammatory reaction, fibroblast proliferation, collagen deposition and tissue remodeling, should lead to the development of effective treatments against pulmonary fibrosis. Evidence that apoptosis plays an important role in the pathophysiology of pulmonary fibrosis has been accumulated. We overview the role of apoptosis in each of the pathogenic events which have emerged from animal models and human tissue studies.
Asunto(s)
Apoptosis , Lesión Pulmonar , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/fisiopatología , Animales , División Celular , Colágeno/metabolismo , Citocinas/metabolismo , Células Endoteliales/patología , Células Epiteliales/patología , Fibroblastos , Sustancias de Crecimiento/metabolismo , Humanos , Inflamación/complicaciones , Inflamación/patología , Modelos Biológicos , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Receptor fasRESUMEN
BACKGROUND: Previous work has shown that all-trans-retinoic acid reverses elastase induced emphysema in rats. Since there is currently no effective treatment for pulmonary emphysema, the effect of retinoic acid should be further investigated in other adult species. A study was undertaken using two murine models of emphysema to evaluate the effect of retinoic acid. METHODS: The models used were an elastase induced emphysema model for acute alveolar destruction and a tumour necrosis factor (TNF)-alpha transgenic mouse which exhibits chronic air space enlargement, loss of elastic recoil, increased lung volume, and pulmonary hypertension comparable to human pulmonary emphysema. All-trans-retinoic acid (2 mg/kg) was injected for 12 successive days after the establishment of emphysema. The effects of treatment were evaluated using physiological and morphometric analyses. RESULTS: In contrast to the rat, administration of all-trans-retinoic acid in these murine models did not improve the emphysema. Moreover, worsening of emphysema was observed in TNF-alpha transgenic mice treated with all-trans-retinoic acid. The level of keratinocyte chemoattractant (KC), a CXC chemokine, in bronchoalveolar lavage fluid was increased in TNF-alpha transgenic mice following retinoic acid treatment. These data raise the possibility that retinoic acid causes deterioration of emphysema by promoting inflammation in this model. CONCLUSIONS: In these models, retinoic acid did not show positive effects on emphysema. The effect of retinoic acid in the treatment of pulmonary emphysema remains controversial, and further studies are required to determine its physiological effects under a variety of experimental conditions.
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Enfisema Pulmonar/tratamiento farmacológico , Tretinoina/uso terapéutico , Animales , Líquido del Lavado Bronquioalveolar/citología , Quimiocinas/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Metaloproteasas/análisis , Ratones , Ratones Endogámicos C57BL , Elastasa Pancreática/toxicidad , Enfisema Pulmonar/patología , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
Lung epithelial cells are a primary target for reactive oxygen species (ROS). ROS can cause oxidative deoxyribonucleic acid modification, such as 8-hydroxy-deoxyguanosine (8-OHdG). A human homologue of the MutT protein (hMTH1) prevents this modification. Mitochondria are the most important cellular source of ROS and may be susceptible to oxidative damage. The purpose of this study is to investigate oxidative stress and mitochondrial damage in lung epithelial cells from idiopathic interstitial pneumonias (IIPs). The authors analysed 8-OHdG, hMTH1, and mitochondrial proteins on lung specimens from 13 patients with IlPs consisted of eight patients with usual interstitial pneumonia and five patients with nonspecific interstitial pneumonia using Western blot analysis and immunohistochemistry. Immunoreactivity for 8-OHdG and hMTH1 was significantly increased in the lung epithelial cells from patients with IIPs compared with controls. The expression of hMTH1 was localised in the nuclear and cytoplasmic, but not the mitochondrial, fraction of lung homogenates. Immunoreactivity for mitochondrial protein and cytochrome c oxidase complex subunit IV was increased in the lung epithelial cells from patients with IIPs compared with controls. The current study concludes that oxidative stress may participate in epithelial cell damage in idiopathic interstitial pneumonia, and that increased mitochondrial mass may associate with increased reactive oxygen species production in idiopathic interstitial pneumonia.
Asunto(s)
Enzimas Reparadoras del ADN , Enfermedades Pulmonares Intersticiales/metabolismo , Pulmón/metabolismo , Estrés Oxidativo , Western Blotting , Desoxiadenosinas/análisis , Complejo IV de Transporte de Electrones/análisis , Células Epiteliales/fisiología , Femenino , Humanos , Inmunohistoquímica , Pulmón/química , Pulmón/patología , Enfermedades Pulmonares Intersticiales/patología , Masculino , Persona de Mediana Edad , Mitocondrias/patología , Proteínas Mitocondriales/análisis , Monoéster Fosfórico Hidrolasas/análisisRESUMEN
The current authors have demonstrated previously that epithelial cell apoptosis, induced by the Fas-Fas ligand pathway, might be involved in fibrosing lung diseases. Whereas lung epithelial cells are sensitive to the Fas-mediated apoptosis, lung fibroblasts may be resistant to Fas-mediated apoptosis and replace damaged epithelial cells. The WI-38 lung fibroblast cell line and primary lung fibroblasts were used to examine the resistant to Fas-mediated apoptosis and the association of anti-apoptotic proteins with this resistance. The administration of agonistic anti-Fas antibody (CH-11) or cycloheximide alone did not induce apoptosis, whereas the co-administration of CH-11 with cycloheximide induced apoptosis in WI-38 cells, in which caspase-8 and -3, but not -9, were activated, and X chromosome-linked inhibitor of apoptosis (ILP) and FLICE-like inhibitor protein (FLIP(L)), but not bcl-xL and bcl-2, were remarkably down regulated. Primary lung fibroblasts were also resistant to Fas-mediated apoptosis, and ILP and FLIP appeared to be involved in this resistance. Furthermore, the results of immunohistochemistry demonstrated that fibroblasts expressed ILP and FLIP(L) proteins in lung tissues from patients with idiopathic pulmonary fibrosis. These results suggest that anti-apoptotic proteins such as X chromosome-linked inhibitor of apoptosis and FLICE-like inhibitor protein may play an important role in preventing Fas-mediated apoptosis in lung fibroblasts, and participate in the development of pulmonary fibrosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Péptidos y Proteínas de Señalización Intracelular , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Fibrosis Pulmonar/fisiopatología , Receptor fas/análisis , Receptor fas/farmacología , Adulto , Anciano , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/análisis , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Línea Celular/efectos de los fármacos , Línea Celular/patología , Línea Celular/fisiología , Inhibidores Enzimáticos/análisis , Fibroblastos/patología , Humanos , Técnicas In Vitro , Proteínas Inhibidoras de la Apoptosis , Pulmón/patología , Persona de Mediana Edad , Proteínas/análisis , Fibrosis Pulmonar/patologíaRESUMEN
BACKGROUND: KL-6, and surfactant protein A (SP-A) and surfactant protein D (SP-D) derived from alveolar type II cells and/or bronchiolar epithelial cells have been reported to be useful markers for interstitial lung diseases. OBJECTIVE: The aim of this study was to measure the levels of these molecules in bronchoalveolar lavage fluid (BALF) from patients with pulmonary sarcoidosis to investigate their relationship with other markers of inflammatory activity. METHODS: We measured KL-6, SP-A and SP-D levels in BALF from patients with pulmonary sarcoidosis using an ELISA. RESULTS: KL-6 and SP-D, but not SP-A levels were significantly increased in pulmonary sarcoidosis compared with controls. KL-6, SP-A and SP-D levels were significantly correlated with each other. KL-6 and SP-D levels were relatively and significantly correlated with the percentage of lymphocytes in BALF. KL-6, SP-D, but not SP-A levels were significantly correlated with the concentration of albumin in BALF. There was no significant correlation between KL-6, SP-A, or SP-D levels and chest X-ray findings, angiotensin-converting enzyme levels, or CD4/CD8 ratio in BALF. CONCLUSIONS: We conclude that KL-6 and SP-D levels in BALF were increased in pulmonary sarcoidosis. Since these markers are specifically derived from epithelial cells, it is considered that KL-6 and SP-D levels are reflecting damage or release of these markers from epithelial cells due to the inflammatory response.
Asunto(s)
Líquido del Lavado Bronquioalveolar/química , Glicoproteínas/metabolismo , Proteolípidos/metabolismo , Surfactantes Pulmonares/metabolismo , Sarcoidosis Pulmonar/metabolismo , Adulto , Anciano , Antígenos , Antígenos de Neoplasias , Biomarcadores/análisis , Líquido del Lavado Bronquioalveolar/citología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Mucina-1 , Mucinas , Proteína A Asociada a Surfactante Pulmonar , Proteína D Asociada a Surfactante Pulmonar , Proteínas Asociadas a Surfactante Pulmonar , Sarcoidosis Pulmonar/epidemiología , Estadística como AsuntoRESUMEN
An antimicrobial protein granulysin is constitutively expressed in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. However, little is known about the precise regulatory mechanisms underlying granulysin gene expression. In this study, we examined the regulatory mechanisms underlying granulysin gene expression using a human monocytic cell line, THP-1, treated with Acholeplasma laidlawii. The level of granulysin mRNA expression in THP-1 cells was significantly augmented in response to stimulation with A. laidlawii. The transfection of reporter gene constructs into THP-1 cells indicated that DNA sequences between residues -329 and -239, relative to the transcriptional start site of the granulysin gene, are responsible for mediating gene induction. In addition, mutagenesis of a putative activator protein-1 (AP-1)-binding site between residues -277 and -271 in the granulysin promoter resulted in the reduction of granulysin promoter activity. Electrophoretic mobility shift assays (EMSA) demonstrated that nuclear extract prepared from A. laidlawii-treated THP-1 cells can generate specific binding to DNA oligonucleotides encompassing the AP-1-binding site, whereas unstimulated nuclear extract from the cells failed to do so. Furthermore, competition and supershift assays confirmed that A. laidlawii can induce the activation of AP-1. These results indicate that AP-1 dominantly participates in the regulation of inducible granulysin gene expression in THP-1 cells. Therefore, the finding of inducible granulysin gene expression by A. laidlawii suggests that inducible granulysin in macrophages may function as a protective weapon when microbial invasion occurs.
Asunto(s)
Acholeplasma laidlawii/inmunología , Antígenos Bacterianos/inmunología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Factor de Transcripción AP-1/inmunología , Regulación hacia Arriba/inmunología , Antígenos de Diferenciación de Linfocitos T/genética , Secuencia de Bases , Sitios de Unión/genética , Sitios de Unión/inmunología , Línea Celular , Humanos , Activación de Macrófagos/inmunología , Datos de Secuencia Molecular , Monocitos/inmunología , Mutación , Regiones Promotoras Genéticas/inmunología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Preischaemic treatment with N-methyl-1-deoxynojirimycin (MOR-14), an alpha-1,6-glucosidase inhibitor, attenuates glycogenolysis and lactate accumulation during ischaemia and markedly reduces infarct size in rabbit hearts. In the present study, we have investigated whether protein kinase C (PKC), a principal mediator of ischaemic preconditioning, is also involved in the cardioprotective effect of MOR-14. To assess the effect of PKC inhibition on infarct size in MOR-14-treated hearts, 38 rabbits were subjected to 30 min of ischaemia followed by 48 h of reperfusion. Infarct size, as a per cent of area at risk, was significantly smaller in rabbits administered 100 mg kg(-1) of MOR-14 10 min before ischaemia (17+/-2%, n=10), than in a control group (46+/-5%, n=10). This beneficial effect of MOR-14 was abolished when 5 mg kg(-1) of chelerythrine, a PKC inhibitor, was given 10 min prior to MOR-14 injection (39+/-4%, n=10), although chelerythrine alone did not alter infarct size (43+/-4%, n=8). Further, chelerythrine had no effect on MOR-14-induced attenuation of glycogen breakdown and lactate accumulation in hearts excised at 30 min of ischaemia. Immunoblot analysis of PKC in homogenates of Langendorff-perfused rabbit hearts revealed that MOR-14 significantly increased levels of PKC-epsilon in the particulate fraction at 20 and 30 min of ischaemia and in the cytosolic fraction at 30 min of ischaemia. Taken as a whole, our data suggest that PKC acts downstream of the inhibition of glycogenolysis by MOR-14 to reduce infarct size. Thus, activation of PKC is a more direct mediator of the cardioprotection afforded by MOR-14 than is inhibition of glycogenolysis.
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1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Glicósido Hidrolasas , Infarto del Miocardio/prevención & control , Proteína Quinasa C/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Glucógeno/metabolismo , Isoenzimas/metabolismo , Lactatos/metabolismo , Masculino , Infarto del Miocardio/patología , Miocardio/metabolismo , Miocardio/patología , Proteína Quinasa C/metabolismo , Conejos , Factores de Tiempo , alfa-Glucosidasas/metabolismoRESUMEN
We previously demonstrated essential roles of the Fas-Fas ligand (FasL) pathway in bleomycin-induced pneumopathy in mice. T lymphocytes and natural killer cells express FasL on activation and use it as a cytotoxic effector molecule. Because interleukin (IL)-12 is known to play a critical role in cell-mediated immunity, we investigated whether anti-IL-12 antibody treatment suppresses the development of this model. The anti-IL-12 antibody treatment decreased the number of apoptotic cells and the degree of inflammation and fibrosis in lung tissue. The results of RT-PCR showed that IL-12p40, IL-12 receptor (R) beta2, interferon-gamma, tumor necrosis factor-alpha and FasL mRNAs were upregulated after bleomycin instillation. The upregulation of FasL, IL-12Rbeta2, and tumor necrosis factor-alpha mRNA expression in lung tissue was suppressed by anti-IL-12 antibody treatment. The results of enzyme-linked immunosorbent assay showed that the levels of IL-12p40, but not of IL-12p70, were increased in lung tissue after bleomycin instillation. Although the increase in IL-12Rbeta2 mRNA levels suggests that the T helper type 1 cell response may participate in lung injury, the increase in IL-12p40 supports T helper type 2 cell predominance in the fibrotic process of this model. The administration of anti-IL-12 antibody could be a novel therapy against lung injury and pulmonary fibrosis.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Interleucina-12/antagonistas & inhibidores , Fibrosis Pulmonar/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Bleomicina , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Recuento de Células , Citocinas/análisis , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Proteína Ligando Fas , Hidroxiprolina/metabolismo , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Interleucina-12/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , ARN Mensajero/metabolismo , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/efectos de los fármacos , Células Th2/inmunología , Células Th2/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
The caspase cascade is an executioner of apoptosis, mediated by Fas. Fas-associating protein with death domain (FADD) interacts with Fas and initiates apoptosis through activating caspase-8. It has previously been demonstrated that the Fas-Fas ligand pathway may be involved in the pathophysiology of idiopathic pulmonary fibrosis (IPF). The aim of this study was to investigate Fas-signalling molecules in epithelial cells in IPF. The immunohistochemistry for FADD and caspase-1 and -3 and terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick endlabelling (TUNEL) methods were performed in lung tissues from 10 patients with IPF obtained by thoracoscopic biopsy and in seven normal lung parenchyma specimens. The induction of caspases expression and activation by Fas-ligation on lung epithelial cell line A549 was also investigated. The immunoreactivity grade for FADD and caspase-1 and -3, and positive signals for TUNEL were significantly increased in epithelial cells of IPF compared with controls. Fas-ligation induced upregulation of caspase-1 and -3 expression in the nucleus and cytoplasm in A549 cells. Procaspase-1, -3, and -8 were activated in apoptotic cells, but not in viable cells. Although direct measurement of the caspase activity in lung epithelial cells of idiopathic pulmonary fibrosis could not be made, these results suggest that the Fas-signalling pathway is upregulated in lung epithelial cells of idiopathic pulmonary fibrosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Proteínas Portadoras/análisis , Pulmón/metabolismo , Fibrosis Pulmonar/metabolismo , Transducción de Señal , Anciano , Western Blotting , Caspasa 1/análisis , Caspasa 3 , Caspasas/análisis , Línea Celular , Activación Enzimática , Células Epiteliales/metabolismo , Proteína de Dominio de Muerte Asociada a Fas , Femenino , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Pulmón/patología , Masculino , Persona de Mediana Edad , Fibrosis Pulmonar/patología , Regulación hacia ArribaRESUMEN
Anthrax toxin lethal factor (LF) in combination with anthrax toxin protective antigen (PA) was endocytosed and translocated to the cytosol of mammalian cells. Residues 1-255 of anthrax toxin lethal factor (LFn) was fused to a cytotoxic T lymphocyte (CTL) epitope of an influenza virus. For processing the toxins, PA must be cleaved into a 63-kDa fragment (PA63) by furin, which is a subtilisin-like processing endo-protease expressed by many eukaryotic cells. To test the ability of cells treated with the LFn fusion protein plus PA to deliver the epitope, CTL assay was performed. Two types of cell lines were identified, one was able to deliver CTL epitope while the other failed to efficiently deliver the epitope. To further elucidate the differences between these cells, the role of furin in these cells was examined. Disruption of the furin gene reduced its ability to deliver the CTL epitope. Furin expression in cells capable of efficiently delivering CTL epitope was quantitatively higher than in cells unable to deliver the epitope. The results suggest that furin plays a critical role in delivery of the CTL epitope of LFn fusion protein.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Antígenos Bacterianos , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/metabolismo , Epítopos de Linfocito T/inmunología , Subtilisinas/metabolismo , Linfocitos T Citotóxicos/inmunología , Animales , Carbunco/inmunología , Vacunas contra el Carbunco/química , Vacunas contra el Carbunco/metabolismo , Antígenos Virales/genética , Antígenos Virales/inmunología , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Western Blotting , Células Cultivadas , Cloroquina/farmacología , Citotoxicidad Inmunológica/efectos de los fármacos , Epítopos de Linfocito T/genética , Femenino , Citometría de Flujo , Furina , Eliminación de Gen , Expresión Génica , Orthomyxoviridae/genética , Orthomyxoviridae/inmunología , Ovalbúmina/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Subtilisinas/genética , TransfecciónRESUMEN
Caspases have been implicated in the effector process of apoptosis in several systems including the Fas-Fas ligand pathway. We previously demonstrated that excessive apoptosis of lung epithelial cells and the Fas-Fas ligand pathway were essential in the pathogenesis of bleomycin-induced pneumopathy in mice. Therefore, the purpose of this study was to investigate whether a caspase inhibitor could prevent the development of this model. The expression of caspase-1 and caspase-3 was upregulated on lung epithelial cells, alveolar macrophages, and infiltrating inflammatory cells in this model. We demonstrated that a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, decreased the caspase-1- and caspase-3-like activity, the number of apoptotic cells, the pathological grade of lung inflammation and fibrosis, and the hydroxyproline content in lung tissues in this model. We conclude that caspase inhibitors could be a new therapeutic approach against lung injury and pulmonary fibrosis.