RESUMEN

Pseudomonas aeruginosa is a frequent cause of antimicrobial-resistant hospital-acquired pneumonia, especially in critically ill patients. Inflammation triggered by P. aeruginosa infection is necessary for bacterial clearance but must be spatially and temporally regulated to prevent further tissue damage and bacterial dissemination. Emerging data have shed light on the pro-resolving actions of angiotensin-(1-7) [Ang-(1-7)] signaling through the G protein-coupled receptor Mas (MasR) during infections. Herein, we investigated the role of the Ang-(1-7)/Mas axis in pneumonia caused by P. aeruginosa by using genetic and pharmacological approach and found that Mas receptor-deficient animals developed a more severe form of pneumonia showing higher neutrophilic infiltration into the airways, bacterial load, cytokines, and chemokines production and more severe pulmonary damage. Conversely, treatment of pseudomonas-infected mice with Ang-(1-7) was able to decrease neutrophilic infiltration in airways and lungs, local and systemic levels of pro-inflammatory cytokines and chemokines, and increase the efferocytosis rates, mitigating lung damage/dysfunction caused by infection. Notably, the therapeutic association of Ang-(1-7) with antibiotics improved the survival rates of mice subjected to lethal inoculum of P. aeruginosa, extending the therapeutic window for imipenem. Mechanistically, Ang-(1-7) increased phagocytosis of bacteria by neutrophils and macrophages to accelerate pathogen clearance. Altogether, harnessing the Ang-(1-7) pathway during infection is a potential strategy for the development of host-directed therapies to promote mechanisms of resistance and resilience to pneumonia.

Asunto(s)

Angiotensina I , Antibacterianos , Ratones Endogámicos C57BL , Fragmentos de Péptidos , Proto-Oncogenes Mas , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Receptores Acoplados a Proteínas G , Animales , Angiotensina I/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Ratones , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Neumonía Bacteriana/tratamiento farmacológico , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patología , Neumonía Bacteriana/metabolismo , Citocinas/metabolismo , Ratones Noqueados , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Neumonía/microbiología , Masculino , Pulmón/microbiología , Pulmón/metabolismo , Pulmón/patología , Transducción de Señal/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos

RESUMEN

Antimicrobial resistance is one of the current public health challenges to be solved. The World Health Organization (WHO) has urgently called for the development of strategies to expand the increasingly limited antimicrobial arsenal. The development of anti-virulence therapies is a viable option to counteract bacterial infections with the possibility of reducing the generation of resistance. Here we report on the chemical structures of pyrrolidones DEXT 1-4 (previously identified as furan derivatives) and their anti-virulence activity on Pseudomonas aeruginosa strains. DEXT 1-4 were shown to inhibit biofilm formation, swarming motility, and secretion of ExoU and ExoT effector proteins. Also, the anti-pathogenic property of DEXT-3 alone or in combination with furanone C-30 (quorum sensing inhibitor) or MBX-1641 (type III secretion system inhibitor) was analyzed in a model of necrosis induced by P. aeruginosa PA14. All treatments reduced necrosis; however, only the combination of C-30 50 µM with DEXT-3 100 µM showed significant inhibition of bacterial growth in the inoculation area and systemic dispersion. In conclusion, pyrrolidones DEXT 1-4 are chemical structures capable of reducing the pathogenicity of P. aeruginosa and with the potential for the development of anti-virulence combination therapies.

Asunto(s)

Antibacterianos , Furanos , Hidrocarburos Halogenados , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Pirrolidinonas , Sistemas de Secreción Tipo III/antagonistas & inhibidores , Animales , Antibacterianos/química , Antibacterianos/farmacología , Furanos/química , Furanos/farmacología , Humanos , Hidrocarburos Halogenados/química , Hidrocarburos Halogenados/farmacología , Ratones , Necrosis , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Pirrolidinonas/química , Pirrolidinonas/farmacología , Percepción de Quorum/efectos de los fármacos , Sistemas de Secreción Tipo III/metabolismo , Factores de Virulencia/metabolismo

RESUMEN

Pathogens phagocytosis and the uptake of apoptotic cells (efferocytosis) are essential macrophages tasks, classically considered as mutually exclusive. Macrophages have been observed to polarize into either pro-inflammatory/microbicidal or anti-inflammatory/efferocytic phenotypes. However, macrophage functions have shown to be more complex. Furthermore, little is known about the regulation of efferocytosis under inflammatory conditions. In this study, we elucidate the modulation of the macrophage efferocytic function during an inflammatory stimulus. We find that bone marrow-derived macrophages (BMDM) are very efficient in engulfing both the bacterial pathogen Pseudomonas aeruginosa and apoptotic cells. BMDM showed a high bactericidal capacity unaffected by the concomitant presence of apoptotic material. Plasticity in macrophage programming, in response to changing environmental cues, may modulate efferocytic capability. In this work, we further show that, after phagocyting and processing Pseudomonas aeruginosa, macrophages highly increase their efferocytic capacity without affecting their phagocytic function. Moreover, we demonstrate that Pseudomonas aeruginosa enhances efferocytosis of these phagocytes through the IL-6 signaling pathway. Our results show that the inflammatory response generated by the bacterial processing enhances these macrophages' capacity to control inflammation through an increased efferocytosis.

Asunto(s)

Apoptosis , Macrófagos/inmunología , Macrófagos/microbiología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Células Cultivadas , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Fagocitosis , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patología

RESUMEN

The purpose of this study was to investigate whether myofibroblast-related fibrosis (scarring) after microbial keratitis was modulated by the epithelial basement membrane (EBM) injury and regeneration. Rabbits were infected with Pseudomonas aeruginosa after epithelial scrape injury and the resultant severe keratitis was treated with topical tobramycin. Corneas were analyzed from one to four months after keratitis with slit lamp photos, immunohistochemistry for alpha-smooth muscle actin (α-SMA) and monocyte lineage marker CD11b, and transmission electron microscopy. At one month after keratitis, corneas had no detectible EBM lamina lucida or lamina densa, and the central stroma was packed with myofibroblasts that in some eyes extended to the posterior corneal surface with damage to Descemet's membrane and the endothelium. At one month, a nest of stromal cells in the midst of the SMA + myofibroblasts in the stroma that were CD11b+ may be fibrocyte precursors to myofibroblasts. At two to four months after keratitis, the EBM fully-regenerated and myofibroblasts disappeared from the anterior 60-90% of the stroma of all corneas, except for one four-month post-keratitis cornea where anterior myofibroblasts were still present in one localized pocket in the cornea. The organization of the stromal extracellular matrix also became less disorganized from two to four months after keratitis but remained abnormal compared to controls at the last time point. Myofibroblasts persisted in the posterior 10%-20% of posterior stroma even at four months after keratitis in the central cornea where Descemet's membrane and the endothelium were damaged. This study suggests that the EBM has a critical role in modulating myofibroblast development and fibrosis after keratitis-similar to the role of EBM in fibrosis after photorefractive keratectomy. Damage to EBM likely allows epithelium-derived transforming growth factor beta (TGFß) to penetrate the stroma and drive development and persistence of myofibroblasts. Eventual repair of EBM leads to myofibroblast apoptosis when the cells are deprived of requisite TGFß to maintain viability. The endothelium and Descemet's membrane may serve a similar function modulating TGFß penetration into the posterior stroma-with the source of TGFß likely being the aqueous humor.

Asunto(s)

Sustancia Propia/patología , Úlcera de la Córnea/patología , Lámina Limitante Posterior/fisiología , Epitelio Corneal/fisiología , Infecciones Bacterianas del Ojo/patología , Infecciones por Pseudomonas/patología , Regeneración/fisiología , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Antígeno CD11b/metabolismo , Lesiones de la Cornea/metabolismo , Lesiones de la Cornea/fisiopatología , Sustancia Propia/metabolismo , Úlcera de la Córnea/metabolismo , Modelos Animales de Enfermedad , Infecciones Bacterianas del Ojo/metabolismo , Femenino , Fibrosis/patología , Inmunohistoquímica , Miofibroblastos/patología , Infecciones por Pseudomonas/metabolismo , Conejos

RESUMEN

High glucose concentration in the airway surface liquid (ASL) is an important feature of diabetes that predisposes to respiratory infections. We investigated the role of alveolar epithelial SGLT1 activity on ASL glucose concentration and bacterial proliferation. Non-diabetic and diabetic rats were intranasally treated with saline, isoproterenol (to increase SGLT1 activity) or phlorizin (to decrease SGLT1 activity); 2 hours later, glucose concentration and bacterial proliferation (methicillin-resistant Sthaphylococcus aureus, MRSA and Pseudomonas aeruginosa, P. aeruginosa) were analyzed in bronchoalveolar lavage (BAL); and alveolar SGLT1 was analyzed by immunohistochemistry. BAL glucose concentration and bacterial proliferation increased in diabetic animals: isoproterenol stimulated SGLT1 migration to luminal membrane, and reduced (50%) the BAL glucose concentration; whereas phlorizin increased the BAL glucose concentration (100%). These regulations were accompanied by parallel changes of in vitro MRSA and P. aeruginosa proliferation in BAL (r = 0.9651 and r = 0.9613, respectively, Pearson correlation). The same regulations were observed in in vivo P. aeruginosa proliferation. In summary, the results indicate a relationship among SGLT1 activity, ASL glucose concentration and pulmonary bacterial proliferation. Besides, the study highlights that, in situations of pulmonary infection risk, such as in diabetic subjects, increased SGLT1 activity may prevent bacterial proliferation whereas decreased SGLT1 activity can exacerbate it.

Asunto(s)

Diabetes Mellitus Experimental/metabolismo , Glucosa/metabolismo , Pulmón/metabolismo , Neumonía Bacteriana/metabolismo , Infecciones por Pseudomonas/metabolismo , Transportador 1 de Sodio-Glucosa/genética , Infecciones Estafilocócicas/metabolismo , Aloxano , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/microbiología , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/microbiología , Broncodilatadores/farmacología , Recuento de Colonia Microbiana , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Regulación de la Expresión Génica , Isoproterenol/farmacología , Pulmón/efectos de los fármacos , Pulmón/microbiología , Pulmón/patología , Masculino , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Florizina/farmacología , Neumonía Bacteriana/complicaciones , Neumonía Bacteriana/genética , Neumonía Bacteriana/patología , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Ratas , Ratas Wistar , Transportador 1 de Sodio-Glucosa/agonistas , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Transportador 1 de Sodio-Glucosa/metabolismo , Infecciones Estafilocócicas/complicaciones , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/patología

RESUMEN

The killing of bacterial pathogens by macrophages occurs via the oxidative burst and bacteria have evolved to overcome this challenge and survive, using several virulence and defense strategies, including antioxidant mechanisms. We show here that the 1-Cys peroxiredoxin LsfA from the opportunistic pathogen Pseudomonas aeruginosa is endowed with thiol-dependent peroxidase activity that protects the bacteria from H(2)O(2) and that this protein is implicated in pathogenicity. LsfA belongs to the poorly studied Prx6 subfamily of peroxiredoxins. The function of these peroxiredoxins has not been characterized in bacteria, and their contribution to host-pathogen interactions remains unknown. Infection of macrophages with the lsfA mutant strains resulted in higher levels of the cytokine TNF-α production due to the activation of the NF-kB and MAPK pathways, that are partially inhibited by the wild-type P. aeruginosa strain. A redox fluorescent probe was more oxidized in the lsfA mutant-infected macrophages than it was in the macrophages infected with the wild-type strain, suggesting that the oxidative burst was overstimulated in the absence of LsfA. Although no differences in the phagocytosis rates were observed when macrophages were infected with wild-type and mutant bacteria in a gentamicin exclusion assay, a higher number of wild-type bacterial cells was found in the supernatant. This difference was not observed when macrophages were pre-treated with a NADPH oxidase inhibitor, confirming the role of LsfA in the bacterial resistance to ROS generated via NADPH oxidase. In an acute pneumonia model, mice infected with the mutant strains presented higher cytokine release in the lungs and increased activated neutrophil recruitment, with reduced bacterial burden and improved survival rates compared to mice infected with the wild-type bacteria. LsfA is the first bacterial 1-Cys Prx shown to modulate host immune responses and its characterization will allow a better understanding of the role of redox signaling in host-pathogen interactions.

Asunto(s)

Interacciones Huésped-Patógeno/inmunología , Macrófagos/inmunología , Peroxirredoxinas/farmacología , Infecciones por Pseudomonas/metabolismo , Animales , Humanos , Peróxido de Hidrógeno/farmacología , Macrófagos/efectos de los fármacos , Ratones , NADPH Oxidasas/metabolismo , Fagocitosis/inmunología , Infecciones por Pseudomonas/virología , Pseudomonas aeruginosa/patogenicidad , Estallido Respiratorio/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Virulencia/efectos de los fármacos

RESUMEN

Pseudomonas aeruginosa is associated with increased mortality in cystic fibrosis (CF) patients, and expresses type III secretion system proteins (TTSP), which is a common mechanism used by gram-negative pathogens for delivery of anti-host factors. Our aim was to investigate whether or not these antigens (TTSP) would be recognized by CF sera, by Western blot reaction. We have showed herein that all patients (n = 11) not chronically infected by P. aeruginosa had their first serum positive for TTSP (ExoS, ExoT, PopB, and/or PopD). All chronic patients had a strong positive serology to TTSP, although relatively weak reactions to TTSP were observed for some individuals in the negative control group. Therefore, TTSP that were early produced in P. aeruginosa infected CF patients, induced a detectable antibody response in those patients and were easily detected by Western-blot reaction.

Asunto(s)

Fibrosis Quística/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Infecciones del Sistema Respiratorio/microbiología , Adolescente , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/sangre , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/sangre , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Niño , Preescolar , Estudios Transversales , Fibrosis Quística/sangre , Fibrosis Quística/metabolismo , Humanos , Infecciones por Pseudomonas/sangre , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/metabolismo , Estudios Retrospectivos

RESUMEN

BACKGROUND: Multi-drug efflux pumps have been increasingly recognized as a major component of resistance in P. aeruginosa. We have investigated the expression level of efflux systems among clinical isolates of P. aeruginosa, regardless of their antimicrobial susceptibility profile. RESULTS: Aztreonam exhibited the highest in vitro activity against the P. aeruginosa isolates studied (64.4% susceptibility), whereas susceptibility rates of imipenem and meropenem were both 47.5%. The MexXY-OprM and MexAB-OprM efflux systems were overexpressed in 50.8% and 27.1% of isolates studied, respectively. Overexpression of the MexEF-OprN and MexCD-OprJ systems was not observed. AmpC beta-lactamase was overexpressed in 11.9% of P. aeruginosa isolates. In addition, decreased oprD expression was also observed in 69.5% of the whole collection, and in 87.1% of the imipenem non-susceptible P. aeruginosa clinical isolates. The MBL-encoding genes blaSPM-1 and blaIMP-1 were detected in 23.7% and 1.7% P. aeruginosa isolates, respectively. The blaGES-1 was detected in 5.1% of the isolates, while blaGES-5 and blaCTX-M-2 were observed in 1.7% of the isolates evaluated. In the present study, we have observed that efflux systems represent an adjuvant mechanism for antimicrobial resistance. CONCLUSIONS: Efflux systems in association of distinct mechanisms such as the porin down-regulation, AmpC overproduction and secondary beta-lactamases play also an important role in the multi-drug resistance phenotype among P. aeruginosa clinical isolates.

Asunto(s)

Bacteriemia/metabolismo , Proteínas Bacterianas/metabolismo , Regulación hacia Abajo , Regulación Bacteriana de la Expresión Génica , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Porinas/metabolismo , Pseudomonas aeruginosa/metabolismo , beta-Lactamasas/metabolismo , Adolescente , Adulto , Antibacterianos/farmacología , Bacteriemia/microbiología , Proteínas Bacterianas/genética , Brasil , Niño , Resistencia a Múltiples Medicamentos , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Porinas/genética , Unión Proteica , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Adulto Joven , beta-Lactamasas/genética

RESUMEN

Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure.

Asunto(s)

Fenómenos Biofísicos , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilcolina/metabolismo , Pseudomonas aeruginosa/enzimología , Catálisis , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Cuerpos de Inclusión/química , Cuerpos de Inclusión/enzimología , Cuerpos de Inclusión/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Fosforilcolina/análisis , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo

RESUMEN

The opportunistic pathogen Pseudomonas aeruginosa PA14 possesses four fimbrial cup clusters, which may confer the ability to adapt to different environments. cupD lies in the pathogenicity island PAPI-1 next to genes coding for a putative phosphorelay system composed of the hybrid histidine kinase RcsC and the response regulator RcsB. The main focus of this work was the regulation of cupD at the mRNA level. It was found that the HN-S-like protein MvaT does not exert a strong influence on cupD transcript levels, as it does for cupA. cupD transcription is higher in cultures grown at 28 degrees C, which agrees with a cupD mutant presenting attenuated virulence only in a plant model, but not in a mouse model of infection. Whereas an rcsC in-frame deletion mutant presented higher levels of cupD mRNA, rcsB deletion had the opposite effect. Accordingly, overexpression of RcsB increased the levels of cupD transcription, and promoted biofilm formation and the appearance of fimbriae. A single transcription start site was determined for cupD and transcription from this site was induced by RcsB. A motif similar to the enterobacterial RcsB/RcsA-binding site was detected adjacent to the -35 region, suggesting that this could be the RcsB-binding site. Comparison of P. aeruginosa and Escherichia coli Rcs may provide insights into how similar systems can be used by different bacteria to control gene expression and to adapt to various environmental conditions.

Asunto(s)

Proteínas Bacterianas/metabolismo , Genes Bacterianos , Pseudomonas aeruginosa/fisiología , Proteínas Represoras/metabolismo , Transcripción Genética , Secuencia de Bases , Biopelículas , Regulación Bacteriana de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Temperatura , Transactivadores/metabolismo

RESUMEN

OBJECTIVE: To evaluate whether respiratory therapy followed by the use of inhaled albuterol modifies the pulmonary deposition of inhaled tobramycin in patients with cystic fibrosis (CF) and whether pulmonary deposition correlates with disease severity or genotype. METHODS: A prospective study was carried out including patients with CF older than 6 years of age and colonized with Pseudomonas aeruginosa. Exclusion criteria were pulmonary exacerbation, changes in therapy between the study phases and FEV1 < 25%. All patients were submitted to pulmonary scintigraphy by means of a scintillation camera equipped with a low energy all purpose collimator in order to evaluate drug penetration following the administration of inhaled 99mTc-tobramycin, as well as to pulmonary perfusion with 99mTc-macroaggregated albumin (phase 1). One month later, the same procedure was performed following respiratory therapy and administration of inhaled albuterol (phase 2). RESULTS: We included 24 patients (12 males) aged 5-27 years (mean +/- SD: 12.85 +/- 6.64 years). The Shwachman score (SS) was excellent/good in 8 patients, moderate/fair in 16 and poor in 0. Genotyping revealed that 7 patients were DeltaF508 homozygotes, 13 were DeltaF508 heterozygotes; and 4 presented other mutations. In all patients, lung deposition of tobramycin decreased in phase 2, especially in those with moderate/fair SS (p = 0.017) and in heterozygotes (p = 0.043). CONCLUSIONS: The use of a respiratory therapy technique and the administration of inhaled albuterol immediately prior to the use of inhaled tobramycin decreased the pulmonary deposition of the latter in CF patients, and this reduction correlates with disease severity and genotype.

Asunto(s)

Antibacterianos/farmacocinética , Fibrosis Quística/terapia , Pulmón/metabolismo , Infecciones por Pseudomonas/metabolismo , Terapia Respiratoria , Tobramicina/farmacocinética , Administración por Inhalación , Adolescente , Adulto , Albuterol/administración & dosificación , Broncodilatadores/administración & dosificación , Niño , Preescolar , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Métodos Epidemiológicos , Femenino , Genotipo , Humanos , Masculino , Mutación , Infecciones por Pseudomonas/tratamiento farmacológico , Radiofármacos/farmacocinética , Tecnecio/farmacocinética , Adulto Joven

RESUMEN

OBJETIVO: Avaliar se a fisioterapia respiratória seguida do uso de salbutamol inalatório modifica a deposição pulmonar de tobramicina inalatória em pacientes com fibrose cística (FC) e se a deposição pulmonar apresenta correlação com a gravidade da doença ou com o genótipo. MÉTODOS: Um estudo prospectivo foi realizado com pacientes com FC maiores de 6 anos e colonizados por Pseudomonas aeruginosa. Os critérios de exclusão foram exacerbação pulmonar, mudança terapêutica entre as fases do estudo e FEV1 < 25 por cento. Todos os pacientes foram submetidos à cintilografia pulmonar com câmara de cintilação com um colimador low energy all purpose para avaliar a penetração da droga após a inalação de tobramicina marcada com tecnécio (99mTc-tobramicina), e à perfusão pulmonar com 99mTc-macroagregados de albumina (fase 1). Após um mês, foi realizado o mesmo procedimento precedido de fisioterapia respiratória e administração de salbutamol inalatório (fase 2). RESULTADOS: Foram incluídos 24 pacientes (12 masculinos) com idade variando de 5 a 27 anos (média ± dp: 12,85 ± 6,64 anos). O escore de Shwachman (ES) foi excelente/bom em 8 pacientes, moderado/regular em 8 e grave em 0. A genotipagem revelou que 7 pacientes eram ΔF508 homozigotos, 13 eram ΔF508 heterozigotos, e 4 apresentavam outras mutações. A deposição pulmonar da tobramicina foi menor na fase 2 em todos os pacientes, sendo menor nos pacientes com ES moderado/regular (p = 0,017) e também nos heterozigotos (p = 0,043). CONCLUSÕES: O uso de uma técnica de fisioterapia respiratória e a administração de salbutamol inalatório imediatamente antes do uso de tobramicina inalada diminuem a deposição pulmonar desta última em pacientes com FC, e esta redução tem correlação com a gravidade da doença e genótipo.

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OBJECTIVE: To evaluate whether respiratory therapy followed by the use of inhaled albuterol modifies the pulmonary deposition of inhaled tobramycin in patients with cystic fibrosis (CF) and whether pulmonary deposition correlates with disease severity or genotype. METHODS: A prospective study was carried out including patients with CF older than 6 years of age and colonized with Pseudomonas aeruginosa. Exclusion criteria were pulmonary exacerbation, changes in therapy between the study phases and FEV1 < 25 percent. All patients were submitted to pulmonary scintigraphy by means of a scintillation camera equipped with a low energy all purpose collimator in order to evaluate drug penetration following the administration of inhaled 99mTc-tobramycin, as well as to pulmonary perfusion with 99mTc-macroaggregated albumin (phase 1). One month later, the same procedure was performed following respiratory therapy and administration of inhaled albuterol (phase 2). RESULTS: We included 24 patients (12 males) aged 5-27 years (mean ± SD: 12.85 ± 6.64 years). The Shwachman score (SS) was excellent/good in 8 patients, moderate/fair in 16 and poor in 0. Genotyping revealed that 7 patients were ΔF508 homozygotes, 13 were ΔF508 heterozygotes; and 4 presented other mutations. In all patients, lung deposition of tobramycin decreased in phase 2, especially in those with moderate/fair SS (p = 0.017) and in heterozygotes (p = 0.043). CONCLUSIONS: The use of a respiratory therapy technique and the administration of inhaled albuterol immediately prior to the use of inhaled tobramycin decreased the pulmonary deposition of the latter in CF patients, and this reduction correlates with disease severity and genotype.

Asunto(s)

Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Adulto Joven , Antibacterianos/farmacocinética , Fibrosis Quística/terapia , Pulmón/metabolismo , Infecciones por Pseudomonas/metabolismo , Terapia Respiratoria , Tobramicina/farmacocinética , Administración por Inhalación , Albuterol/administración & dosificación , Broncodilatadores/administración & dosificación , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Métodos Epidemiológicos , Genotipo , Mutación , Infecciones por Pseudomonas/tratamiento farmacológico , Radiofármacos , Radiofármacos/farmacocinética , Tecnecio , Tecnecio/farmacocinética , Adulto Joven

RESUMEN

This report addressed the question whether ExoU stimulation of airway epithelial cells may contribute to the inflammatory response detected in the course of Pseudomonas aeruginosa respiratory infections. Infection with PA103 P. aeruginosa elicited a potent release of IL-6 and IL-8, as well as of arachidonic acid (AA) and PGE(2) that was reduced by the bacterial treatment with MAFP, a cPLA(2) inhibitor. Airway cells from the BEAS-2B line and in primary culture were shown to be enriched in lipid bodies (LBs), that are cytoplasmic domains implicated in AA transformation into eicosanoids. However, cells infected with PA103 and with a mutant deficient in exoU but complemented with a functional gene exhibited reduced contents of LBs, and this reduction was inhibited by MAFP. FACS analysis showed that the decrease in the LB content correlated with the presence of intracellular PGE(2). Also, in PA103-infected cells, PGE(2) was immunolocalized in LBs, suggesting that the reduction in the cell content of the organelles was due to consumption of their glycerolipids, resulting in local synthesis of the prostanoid. In conclusion, we showed the ExoU ability to induce airway epithelial cells to overproduce PGE(2) and we speculate that LB may represent intracellular loci involved in ExoU-induced eicosanoid synthesis.

Asunto(s)

Proteínas Bacterianas/metabolismo , Bronquios/metabolismo , Células Epiteliales/metabolismo , Cuerpos de Inclusión/metabolismo , Mediadores de Inflamación/metabolismo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/farmacología , Proteínas Bacterianas/genética , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/microbiología , Línea Celular , Citocinas/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Humanos , Metabolismo de los Lípidos , Organofosfonatos/farmacología , Prostaglandinas E/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos

RESUMEN

As Pseudomonas aeruginosa ExoU possesses two functional blocks of homology to calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)), we addressed the question whether it would exhibit a proinflammatory activity by enhancing the synthesis of eicosanoids by host organisms. Endothelial cells from the HMEC-1 line infected with the ExoU-producing PA103 strain exhibited a potent release of arachidonic acid (AA) that could be significantly inhibited by methyl arachidonyl fluorophosphonate (MAFP), a specific PLA(2) inhibitor, as well as significant amounts of the cyclooxygenase (COX)-derived prostaglandins PGE(2) and PGI(2). Cells infected with an isogenic mutant defective in ExoU synthesis did not differ from non-infected cells in the AA release and produced prostanoids in significantly lower concentrations. Infection by PA103 induced a marked inflammatory response in two different in vivo experimental models. Inoculation of the parental bacteria into mice footpads led to an early increase in the infected limb volume that could be significantly reduced by inhibitors of both COX and lipoxygenase (ibuprofen and NDGA respectively). In an experimental respiratory infection model, bronchoalveolar lavage (BAL) from mice instilled with 10(4) cfu of PA103 exhibited a marked influx of inflammatory cells and PGE(2) release that could be significantly reduced by indomethacin, a non-selective COX inhibitor. Our results suggest that ExoU may contribute to P. aeruginosa pathogenesis by inducing an eicosanoid-mediated inflammatory response of host organisms.

Asunto(s)

Eicosanoides/biosíntesis , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiología , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Ácido Araquidónico/antagonistas & inhibidores , Ácido Araquidónico/metabolismo , Ácidos Araquidónicos/farmacología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Línea Celular , Dinoprostona/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Epoprostenol/metabolismo , Femenino , Fosfolipasas A2 Grupo IV , Humanos , Ibuprofeno/uso terapéutico , Indometacina/uso terapéutico , Inflamación/patología , Inhibidores de la Lipooxigenasa/uso terapéutico , Masoprocol/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Organofosfonatos/farmacología , Fosfolipasas A/antagonistas & inhibidores , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad

RESUMEN

The mammalian innate immune system recognizes pathogen-associated molecular patterns through pathogen recognition receptors. Nod1 has been described recently as a cytosolic receptor that detects specifically diaminopimelate-containing muropeptides from Gram-negative bacteria peptidoglycan. In the present study we investigated the potential role of Nod1 in the innate immune response against the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that Nod1 detects the P. aeruginosa peptidoglycan leading to NF-kappaB activation and that this activity is diminished in epithelial cells expressing a dominant-negative Nod1 construct or in mouse embryonic fibroblasts from Nod1 knock-out mice infected with P. aeruginosa. Finally, we demonstrate that the cytokine secretion kinetics and bacterial killing are altered in Nod1-deficient cells infected with P. aeruginosa in the early stages of infection.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/fisiología , Apoptosis , Peptidoglicano/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/patogenicidad , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Citocinas/metabolismo , Células Epiteliales/citología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Genes Dominantes , Humanos , Inmunidad Innata , Cinética , Ratones , Ratones Noqueados/microbiología , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD1 , Infecciones por Pseudomonas/metabolismo

RESUMEN

The pharmacokinetics of tobramycin in adolescents or young adults with cystic fibrosis and in age-matched controls were prospectively compared. Patients with CF had a higher tobramycin total body clearance (121.2 +/- 14.2 ml/min/1.73 m2) than did controls (102.2 +/- 18.9 ml/min/1.73 m2, P less than 0.05). This was not associated with a higher glomerular filtration rate (iothalamate total body clearance 147.5 +/- 29.2 ml/min/1.73 m2 in patients vs 142.9 +/- 33.3 ml/min/1.73 m2 in controls) or a lower binding of gentamicin to serum proteins (14.3% +/- 2.6% in patients vs 17.4% +/- 3.8% in controls). Tobramycin renal clearance was not significantly different in the two groups (89.5 +/- 17.9 ml/min/1.73 m2 in patients vs 81.0 +/- 15.8 ml/min/1.73 m2 in controls). In the control group, tobramycin total body and renal clearances were highly correlated with iothalamate total body clearance (r = +0.95 and +0.88, P less than 0.01). In patients with cystic fibrosis, the correlation was not significant (r = +0.56, P greater than 0.05 for total body clearance, and r = 0.32, P greater than 0.1 for renal clearance). There was no significant difference in volume of distribution normalized to body surface area or in half-life of elimination. The higher tobramycin total body clearance without an increase in renal clearance, and the lower correlation with glomerular filtration rate indicate that an extrarenal clearance pathway might play a significant role in the elimination of tobramycin from the serum of patients with cystic fibrosis.

Asunto(s)

Fibrosis Quística/metabolismo , Infecciones por Pseudomonas/metabolismo , Tobramicina/metabolismo , Adolescente , Adulto , Superficie Corporal , Niño , Ensayos Clínicos como Asunto , Femenino , Gentamicinas/metabolismo , Tasa de Filtración Glomerular/efectos de los fármacos , Humanos , Cinética , Masculino , Estudios Prospectivos , Unión Proteica , Infecciones por Pseudomonas/tratamiento farmacológico