Investigating intracellular persistence of Staphylococcus aureus within a murine alveolar macrophage cell line.

OBJECTIVE: Staphylococcus aureus is a particularly difficult pathogen to eradicate from the respiratory tract. Previous studies have highlighted the intracellular capacity of S.aureus in several phagocytic and non-phagocytic cells. The aim of this study was to define S.aureus interaction within a murine alveolar macrophage cell line. METHODS: Cell line MH-S was infected with Newman strain. Molecular mechanisms involved in phagocytosis were explored. To assess whether S.aureus survives intracellularly quantitative (gentamicin protection assays and bacterial plating) and qualitative analysis (immunofluorescence microscopy) were performed. Bacterial colocalization with different markers of the endocytic pathway was examined to characterize its intracellular trafficking. RESULTS: We found that S.aureus uptake requires host actin polymerization, microtubule assembly and activation of phosphatidylinositol 3-kinase signaling. Time course experiments showed that Newman strain was able to persist within macrophages at least until 28.5 h post infection. We observed that intracellular bacteria are located inside an acidic subcellular compartment, which co-localizes with the late endosome/lysosome markers Lamp-1, Rab7 and RILP. Colocalization counts with TMR-dextran might reflect a balance between bacterial killing and intracellular survival. CONCLUSIONS: This study indicates that S.aureus persists and replicates inside murine alveolar macrophages, representing a privileged niche that can potentially offer protection from antimicrobial activity and immunological host defense mechanisms.

Repercusión clínica de las pérdidas de medicación en los equipos de sueroterapia. Experiencia con el paracetamol endovenoso / Clinical impact of failure to administer medication left inside intravenous infusion material: a study of acetaminophen loss

Objetivo: Conocer el método de administración intermitente de un fármaco endovenoso en un servicio de urgencias (SU) y si la retención de fármaco en el equipo de sueroterapia, una vez finalizada la perfusión, repercute sobre la respuesta clínica. Método: Estudio prospectivo, observacional y descriptivo en una primera fase. En una segunda fase ha sido intervencionista y analítico. Fase 1: análisis de la técnica de perfusión y su repercusión en la respuesta terapéutica sobre el paciente. Fase 2: análisis de la técnica de perfusión intermitente tras acción docente de mejora dirigida al personal de enfermería y su repercusión en la respuesta terapéutica sobre el paciente. Fármaco analizado: Paracetamol 1 g/ev. Variables: edad, sexo, peso, minutos de perfusión, volumen residual post-perfusión en los equipos, escala de dolor y/o temperatura basal (15’, 60’y 4 horas), concentración plasmática de paracetamol a las 4 horas. Población de estudio: Individuos de ambos sexos mayores de 15 años de edad que acuden al SU y que se les prescribe paracetamol 1 g/ev. Resultados: Se incluye 119 pacientes, 60 durante la 1ª fase y 59 en la 2ª fase. Ninguna perfusión considerada administrada en su totalidad había sido purgada durante la fase1. El tiempo de administración de la perfusión fue de 25,7 ± 10,9 minutos. La omisión de purga conllevó un volumen (..) (AU)

Objectives: To determine whether acetaminophen is retained inside intravenous infusion bottels and lives after intermittent administration of fluids in the emergency department and whether such retention has an effect on outcomes. Methods: Prospective, observational study in the first phase, followed by a second phase to analyze the effect of technical instruction and intervention. In the first phase, the completeness of intravenous administration of medication and the patient’s response to therapy were recorded. In the second phase, after instruction to improve the staff’s technical performance of intravenous infusion of medication, completeness of administration and patient response were again recorded. The medication studied was acetaminophen (1 g) infused through an intravenous line. Variables recorded were age, sex, weight, duration of infusion, residual volume left in the intravenous infusion equipment, score on a pain scale and/or temperature (at 15 minutes, 1 hour, and 4 hours), and plasma concentration of acetaminophen at 4 hours. The study population consisted of male and female emergency department patients aged 15 years or older who were prescribed 1 g of intravenous acetaminophen. Results: A total of 119 patients were enrolled; 60 were studied in the first phase and 59 in the second. In the first phase, the infusion equipment failed to draim completely in all cases. Intravenous administration took a mean (SD) of 25.7(10.9) minutes during this phase and omission of a flush maneuver at the end led to leaving a mean residual volume of12.65 (2.95) mL in the system. The mean plasma concentration of acetaminophen in the first phase (no instruction to flush the line) was 4.28 (5.04) mg/ìL; in the second phase, after the staff had been instructed to flush the system, the mean plasma concentration was 5,27 (..) (AU)

Lipopolysaccharide-binding protein and CD14 are increased in the bronchoalveolar lavage fluid of smokers.

Lipopolysaccharide-binding protein (LBP) and CD14 contribute to the recognition of pathogens by cells, which triggers the activation of defence responses. Smoking is a risk factor for the development of chronic obstructive pulmonary disease (COPD) and respiratory infections. The current authors theorised that levels of LBP and CD14 in the lungs of smokers would be higher than those in the lungs of never-smokers. These elevated levels could affect host responses upon infection. LBP, soluble CD14 (sCD14) and interleukin (IL)-8 were detected by ELISA. Nuclear factor (NF)-kappaB, p38 and the inhibitor IkappaBalpha were studied by immunoassays. Gene expression was assessed by RT-PCR. Bronchoalveolar lavage levels of LBP and CD14 were significantly higher in smokers and COPD patients than in never-smokers, whereas levels of both proteins were not significantly different between smokers and COPD patients. IL-6, IL-1beta and cigarette smoke condensate induced the expression of LBP and CD14 by airway epithelial cells. LBP and sCD14 inhibited the nontypeable Haemophilus influenzae (NTHi)-dependent secretion of IL-8 and the activation of NF-kappaB and p38 mitogen-activated protein kinase signalling pathways but they increased the internalisation of NTHi by airway epithelial cells. Thus, in the inflamed airways of smokers both proteins could contribute to inhibit bacteria-dependent cellular activation without compromising the internalisation of pathogens by airway cells.

Blunted gamma delta T-lymphocyte response in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterised by an excessive inflammatory response to inhaled particles, mostly tobacco smoking. Although inflammation is present in all smokers, only a percentage of them develop COPD. T-lymphocytes are important effector and regulatory cells that participate actively in the inflammatory response of COPD. They comprise the T-cell receptor (TCR)-alpha beta (CD4+ and CD8+) and TCR-gamma delta T-lymphocytes. The latter represent a small percentage of the total T-cell population, but play a key role in tissue repair and mucosal homeostasis. To investigate TCR-alpha beta (CD4+ and CD8+) and TCR-gamma delta T-lymphocytes in COPD, the present authors determined, by flow cytometry, the distribution of both subpopulations in peripheral blood and bronchoalveolar lavage (BAL) samples obtained from patients with COPD, smokers with normal lung function and never-smokers. The present study found that: 1) the distribution of CD4+ and CD8+ lymphocytes in blood and BAL was similar in all three groups; 2) compared with nonsmokers, gamma delta T-lymphocytes were significantly increased in smokers with preserved lung function; and 3) this response was blunted in patients with COPD. These results highlight a novel, potentially relevant, pathogenic mechanism in chronic obstructive pulmonary disease.