RESUMEN
BACKGROUND: MicroRNA (miRNA) control gene transcription by binding to and repressing the translation of messenger RNA (mRNA). Their role in the acute respiratory distress syndrome (ARDS) is undefined. METHODS: Blood leukocytes from 51 patients enrolled in a prior randomized trial of corticosteroids for ARDS were analyzed. After screening eight patients with microarrays for altered miRNA expression, 25 miRNAs were selected for further analysis using RT-PCR in all 51 patients. RESULTS: On day 0, the 51 patients had APACHE III score of 60.4â±â17.7 and PaO2/FiO2 of 117â±â49. 21 miRNA were expressed at increased levels in blood leukocytes at the onset of ARDS compared with healthy controls. These miRNA remained elevated at day 3 and increased further by day 7 (log2 fold change from 0.66 to 5.7 fold, Pâ<0.05 compared to day 0). In a subgroup analysis (37 patients treated with corticosteroids and 14 treated with placebo), the interaction of miRNA expression over time and steroid administration was not significant suggesting that systemic corticosteroids had no effect on the miRNA detected in our study. In contrast, corticosteroids but not placebo decreased IL-6 and C-reactive protein at day 3 (Pâ<â0.001) demonstrating an early systemic anti-inflammatory response whereas both treatment arms had decreased values by day 7 (Pâ<0.001). CONCLUSIONS: Expression of miRNA is increased in blood leukocytes of patients with ARDS at day 0 and day 3 and rises further by day 7, when systemic inflammation is subsiding. These effects appear independent of the administration of steroids, suggesting different inflammatory modifying roles for each in the resolving phases of ARDS.
Asunto(s)
Leucocitos/metabolismo , MicroARNs/metabolismo , Síndrome de Dificultad Respiratoria/genética , APACHE , Corticoesteroides/uso terapéutico , Adulto , Anciano , Proteína C-Reactiva/metabolismo , Femenino , Humanos , Interleucina-6/metabolismo , Masculino , MicroARNs/genética , Persona de Mediana Edad , Ensayos Clínicos Controlados Aleatorios como Asunto , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Acinetobacter baumannii is a common nosocomial pathogen and strain-typing methods play an important role in hospital outbreak investigations and epidemiologic surveillance. We describe a method for identifying strain-specific peptide markers based on LC-MS/MS profiling of digested peptides. This method classified a test set of A. baumannii isolates collected from a hospital outbreak with discriminatory performance exceeding that of MALDI-TOF mass spectrometry. METHODS: Following the construction of a species "pan-peptidome" by in silico translation and digestion of whole genome sequences, a hypothetical set of genome-specific peptides for an isolate was constructed from the disjoint set of the pan-peptidome and the isolate's calculated peptidome. The genome-specific peptidome guided selection of highly expressed genome-specific peptides from LC-MS/MS experimental profiles as potential peptide markers. The species specificity of each experimentally identified genome-specific peptide was confirmed through a Unipept lowest common ancestor analysis. RESULTS: Fifteen A. baumannii isolates were analyzed to derive a set of genome- and species-specific peptides that could be used as peptide markers. Identified peptides were cross-checked with protein BLAST against a set of 22 A. baumannii whole genome sequences. A subset of these peptide markers was confirmed to be present in the actual peptide profiles generated by multiple reaction monitoring and targeted LC-MS/MS. The experimentally identified peptides separated these isolates into 6 strains that agreed with multilocus sequence typing analysis performed on the same isolates. CONCLUSIONS: This approach may be generalizable to other bacterial species, and the peptides may be useful for rapid MS strain tracking of isolates with broad application to infectious disease diagnosis.
Asunto(s)
Acinetobacter baumannii/química , Acinetobacter baumannii/clasificación , Técnicas de Tipificación Bacteriana/métodos , Proteómica , Espectrometría de Masas en Tándem , Acinetobacter baumannii/aislamiento & purificación , Cromatografía Liquida , Humanos , Péptidos/genética , Péptidos/metabolismoRESUMEN
Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45-, CD34dim that is comparable to a previously described CD45-, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45-, CD34dim, 7-AAD- CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and von Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45-, CD34dim, and 7-AAD- have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification.
Asunto(s)
Antígenos CD34/metabolismo , Separación Celular , Células Endoteliales/citología , Antígenos Comunes de Leucocito/metabolismo , Adulto , Antígeno CD146/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Supervivencia Celular , Dactinomicina/análogos & derivados , Dactinomicina/sangre , Citometría de Flujo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Indoles/química , Leucocitos Mononucleares/citología , Microscopía , Microscopía Fluorescente , Persona de Mediana Edad , Fenotipo , Proyectos Piloto , Lectinas de Plantas/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
OBJECTIVE: Evaluate the effects of methylprednisolone on markers of inflammation, coagulation, and angiogenesis during early acute respiratory distress syndrome. DESIGN: Retrospective analysis. SETTING: Four intensive care units. SUBJECTS: Seventy-nine of 91 patients with available samples enrolled in a randomized, blinded controlled trial. INTERVENTIONS: Early methylprednisolone infusion (n = 55) compared with placebo (n = 24). MEASUREMENTS AND MAIN RESULTS: Interleukin-6, tumor necrosis factor α, vascular endothelial growth factor, protein C, procalcitonin, and proadrenomedullin were measured in archived plasma. Changes from baseline to day 3 and day 7 were compared between groups and in subgroups based on the precipitating cause of acute respiratory distress syndrome. Methylprednisolone therapy was associated with greater improvement in Lung Injury Score (p = .003), shorter duration of mechanical ventilation (p = .005), and lower intensive care unit mortality (p = .05) than control subjects. On days 3 and 7, methylprednisolone decreased interleukin-6 and increased protein C levels (all p < .0001) compared with control subjects. Proadrenomedullin levels were lower by day 3 with methylprednisolone treatment (p = .004). Methylprednisolone decreased interleukin-6 by days 3 and 7 in patients with pulmonary causes of acute respiratory distress syndrome but only at day 3 in those with extrapulmonary causes of acute respiratory distress syndrome. Protein C levels were increased with methylprednisolone on days 3 and 7 in patients with infectious and/or pulmonary causes of acute respiratory distress syndrome (all p < .0001) but not in patients with noninfectious or extrapulmonary causes of acute respiratory distress syndrome. Proadrenomedullin levels were decreased with methylprednisolone on day 3 in patients with infectious or extrapulmonary causes of acute respiratory distress syndrome (both p ≤ .008) but not in noninfectious or pulmonary acute respiratory distress syndrome. Tumor necrosis factor, vascular endothelial growth factor, and procalcitonin were elevated but not differentially affected by methylprednisolone therapy. CONCLUSIONS: In early acute respiratory distress syndrome, administration of methylprednisolone was associated with improvement in important biomarkers of inflammation and coagulation and clinical outcomes. Biomarker changes varied with the precipitating cause of acute respiratory distress syndrome, suggesting that the underlying mechanisms and response to anti-inflammatory therapy may vary with the cause of acute respiratory distress syndrome.
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Coagulación Sanguínea/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Metilprednisolona/administración & dosificación , Neovascularización Fisiológica/efectos de los fármacos , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Coagulación Sanguínea/fisiología , Cuidados Críticos/métodos , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Esquema de Medicación , Diagnóstico Precoz , Femenino , Estudios de Seguimiento , Mortalidad Hospitalaria , Humanos , Mediadores de Inflamación/análisis , Infusiones Intravenosas , Interleucina-6/metabolismo , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Valores de Referencia , Síndrome de Dificultad Respiratoria/diagnóstico , Síndrome de Dificultad Respiratoria/metabolismo , Síndrome de Dificultad Respiratoria/mortalidad , Medición de Riesgo , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Tasa de Supervivencia , Factores de Tiempo , Resultado del TratamientoRESUMEN
To define gene expression profiles that occur during the initial activation of human innate immunity, we administered intravenous endotoxin (n = 8) or saline (n = 4) to healthy subjects and hybridized RNA from blood mononuclear cells (0, 0.5, 6, 24, 168 h) or whole blood (0, 3, 6, 24, 168 h) to oligonucleotide probe arrays. The greatest change in mononuclear cell gene expression occurred at 6 h (439 induced and 428 repressed genes, 1% false discovery rate, and 50% fold change) including increased expression of genes associated with pathogen recognition molecules and signaling cascades linked to receptors associated with cell mobility and activation. Induced defense response genes included cytokines, chemokines, and their respective receptors, acute-phase transcription factors, proteases, arachidonate metabolites, and oxidases. Repressed defense response genes included those associated with co-stimulatory molecules, T and cytotoxic lymphocytes, natural killer (NK) cells, and protein synthesis. Gene expression profiles of whole blood had similar biological themes. Over 100 genes not typically associated with acute inflammation were differentially regulated after endotoxin. By 24 h, gene expression had returned to baseline values. Thus the inflammatory response of circulating leukocytes to endotoxin in humans is characterized by a rapid amplification and subsidence of gene expression. These results indicate that a single intravascular exposure to endotoxin produces a large but temporally short perturbation of the blood transcriptome.
Asunto(s)
Endotoxemia/inmunología , Endotoxinas/toxicidad , Regulación de la Expresión Génica , Inmunidad Innata , Leucocitos Mononucleares/metabolismo , Adulto , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Endotoxemia/sangre , Femenino , Humanos , Inmunidad Innata/genética , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Systemic inflammation because of sepsis results in endothelial cell activation and microvascular injury. High-mobility group protein-1 (HMGB1), a novel inflammatory molecule, is a late mediator of endotoxin shock and is present in the blood of septic patients. The receptor for advanced glycation end products (RAGE) is expressed on endothelium and is a receptor for HMGB1. Here we examine the effects of HMGB1 on human endothelial cell function. Recombinant human HMGB1 (rhHMGB1) was cloned and expressed in Escherichia coli and incubated with human microvascular endothelium. rhHMGB1 caused a dose- and time-dependent increase in the expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and RAGE. rhHMGB1 induced the secretion of tumor necrosis factor-alpha (TNFalpha), interleukin 8 (IL-8), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), and tissue plasminogen activator (tPA) (P <.01). rhHMGB1 stimulation resulted in transient phosphorylation of mitogen-activated protein (MAP) kinases, extracellular signal-related kinase (ERK), Jun N-terminal kinase (JNK), and p38, and in nuclear translocation of transcription factors NF-kappaB and Sp1. These effects are partially mediated by TNFalpha autocrine stimulation, as anti-TNFalpha antibodies significantly decrease chemokine and adhesion molecule responses (P =.002). Thus, rhHMGB1 elicits proinflammatory responses on endothelial cells and may contribute to alterations in endothelial cell function in human inflammation.