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Background Linezolid is recommended for treatment of pneumonia and other invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA). The premise underlying this recommendation is that linezolid inhibits in vivo production of potent staphylococcal exotoxins, including Panton-Valentine leukocidin (PVL) and α-hemolysin (Hla), although supporting evidence is lacking.Methods A rabbit model of necrotizing pneumonia using MRSA clone USA300 was used to compare therapeutic effects of linezolid (50 mg/kg 3 times/day) and vancomycin (30 mg/kg 2 times/day) administered 1.5, 4, and 9 hours after infection on host survival outcomes and in vivo bacterial toxin production.Results Mortality rates were 100% for untreated rabbits and 83%-100% for vancomycin-treated rabbits. In contrast, mortality rates were 25%, 50%, and 100% for rabbits treated with linezolid 1.5, 4, and 9 hours after infection, respectively. Compared with untreated and vancomycin-treated rabbits, improved survival of rabbits treated 1.5 hours after infection with linezolid was associated with a significant decrease in bacterial counts, suppressed bacterial production of PVL and Hla, and reduced production of the neutrophil-chemoattractant interleukin 8 in the lungs.Conclusions Across the study interval, only early treatment with linezolid resulted in significant suppression of exotoxin synthesis and improved survival outcomes in a rabbit model of MRSA necrotizing pneumonia.

Introduction Acute lung injury (ALI), including its more severe form, acute respiratory distress syndrome (ARDS), is common in critically ill patients and carries an overall mortality of 40% or higher [...], Acute lung injury (ALI) is a life-threatening condition in critically ill patients. Injury to the alveolar epithelium is a critical event in ALI, and accumulating evidence suggests that it is linked to proapoptotic Fas/FasL signals. Active soluble FasL (sFasL) is detectable in the bronchoalveolar lavage (BAL) fluid of patients with ALI, but the mechanisms controlling its bioactivity are unclear. We therefore investigated how the structure of sFasL influences cellular activation in human and mouse lungs and the role of oxidants and proteases in modifying sFasL activity. The sFasL in BAL fluid from patients with ALI was bioactive and present in high molecular weight multimers and aggregates. Oxidants generated from neutrophil myeloperoxidase in BAL fluid promoted aggregation of sFasL in vitro and in vivo. Oxidation increased the biological activity of sFasL at low concentrations but degraded sFasL at high concentrations. The amino-terminal extracellular stalk region of human sFasL was required to induce lung injury in mice, and proteolytic cleavage of the stalk region by MMP-7 reduced the bioactivity of sFasL in human cells in vitro. The sFasL recovered from the lungs of patients with ALI contained both oxidized methionine residues and the stalk region. These data provide what we believe to be new insights into the structural determinants of sFasL bioactivity in the lungs of patients with ALI.

Severe infection with respiratory syncytial virus (RSV) in children can progress to respiratory distress and acute lung injury (ALI). Accumulating evidence suggests that mechanical ventilation (MV) is an important cofactor in the development of ALI by modulating the host immune responses to bacteria. This study investigates whether MV enhances the host response to pneumonia virus of mice (PVM), a mouse pneumovirus that has been used as a model for RSV infection in humans. BALB/c mice were inoculated intranasally with diluted clarified lung homogenates from mice infected with PVM strain J3666 or uninfected controls. Four days after inoculation, the mice were subjected to 4 h of MV (tidal volume, 10 ml/kg) or allowed to breathe spontaneously. When compared with that of mice inoculated with PVM only, the administration of MV to PVM-infected mice resulted in increased bronchoalveolar lavage fluid concentrations of the cytokines macrophage inflammatory protein (MIP)-2, MIP-1[alpha] (CCL3), and IL-6; increased alveolar-capillary permeability to high molecular weight proteins; and increased caspase-3 activity in lung homogenates. We conclude that MV enhances the activation of inflammatory and caspase cell death pathways in response to pneumovirus infection. We speculate that MV potentially contributes to the development of lung injury in patients with RSV infection. respiratory syncytial virus; acute lung injury

Acute lung injury in humans is characterized histopathologically by neutrophilic alveolitis, injury of the alveolar epithelium and endothelium, hyaline membrane formation, and microvascular thrombi. Different animal models of experimental lung injury have been used to investigate mechanisms of lung injury. Most are based on reproducing in animals known risk factors for ARDS, such as sepsis, lipid embolism secondary to bone fracture, acid aspiration, ischemia-reperfusion of pulmonary or distal vascular beds, and other clinical risks. However, none of these models fully reproduces the features of human lung injury. The goal of this review is to summarize the strengths and weaknesses of existing models of lung injury. We review the specific features of human ARDS that should be modeled in experimental lung injury and then discuss specific characteristics of animal species that may affect the pulmonary host response to noxious stimuli. We emphasize those models of lung injury that are based on reproducing risk factors for human ARDS in animals and discuss the advantages and disadvantages of each model and the extent to which each model reproduces human ARDS. The present review will help guide investigators in the design and interpretation of animal studies of acute lung injury. acute respiratory distress syndrome; human lung injury

We examined the role of Toll-like receptor (TLR)-4 in modifying the lung inflammatory response and its effects on the bacterial recovery from the lungs following inhaled Escherichia coli in two different strains of TLR-4 mutant mice that are hyporesponsive to LPS. The C57BL/ 10ScN([tlr4.sup.lps-del]) mice containing a deletion mutation in the TLR-4 gene showed lower proinflammatory cytokine levels, lower lung MPO activity, and less parenchymal and peribronchial inflammation compared with the C57BL/10ScSn mice, a related TLR-4 wild-type substrain. However, the C57BL/10ScN([tlr4.sup.lps-del]) mutant showed lower bacterial recovery in the lungs following inhaled E. coli associated with a rapid but transient increase in air space neutrophil counts at 6 h. In comparison, the C3H/HeJ([tlr4.sup.Lps-d]) mutant mice containing a Pro712His substitution in TLR-4 demonstrated lower proinflammatory cytokine levels, lower lung MPO activity, and lower neutrophil accumulation in the air spaces but showed no differences in the bacterial burden of inhaled E. coli at 6 h, when compared with the TLR-4 wild-type C3H/HeSnJ mice. Thus two different TLR-4 mutants showed attenuated inflammatory responses in the lungs, but the reduced inflammatory responses were not consistently associated with either improved or impaired bacterial elimination from the lungs. Our findings indicate that the inflammatory response to inhaled E. coli is TLR-4 dependent, but bacterial elimination depends on other factors in addition to TLR-4. lipopolysaccharide; Toll-like receptor-4; Escherichia coli; bacterial proliferation

Previous animal studies have identified a role for activation of innate immunity in the pathogenesis of ventilator-associated lung injury. These studies have used large tidal volume ventilation to study the effect of alveolar overdistension on induction of inflammatory pathways. We hypothesized an alternative mechanism for the pathogenesis of lung injury in which moderate tidal volume ventilation does not independently cause clinical inflammation but rather interacts with innate immune activation by bacterial products, resulting in an enhanced inflammatory response. We measured cytokine expression and lung injury in normal and lipopolysaccharide (LPS)-treated anesthetized rabbits randomized to either spontaneous respiration or mechanical ventilation. Outcome parameters were analyzed by two-way factorial analysis of variance to identify synergism between ventilation and systemic LPS. Mechanical ventilation alone resulted in minimal cytokine expression in the lung but did enhance LPS-induced expression of tumor necrosis factor-[alpha], the CXC chemokines interleukin-8 and growth-related protein-[alpha] and the CC chemokine monocyte chemoattractant protein-1. Increased mRNA expression and activation of the transcription factors nuclear factor-[kappa]B and activator protein-I accompanied the cytokine responses. We conclude that moderate volume ventilation strategies augment the innate immune response to bacterial products in the lung and may play a role in the development of acute lung injury in patients with sepsis. acute lung injury; innate immunity; transcription factor-1; nuclear factor-[kappa]B

Abstract Background Exposure to mechanical ventilation enhances lung injury in response to various stimuli, such as bacterial endotoxin (LPS). The Fas/FasL system is a receptor ligand system that has dual pro-apoptotic and pro-inflammatory functions and has been implicated in the pathogenesis of lung injury. In this study we test the hypothesis that a functioning Fas/FasL system is required for the development of lung injury in mechanically ventilated mice. Methods C57BL/6 (B6) and Fas-deficient lpr mice were exposed to either intra-tracheal PBS followed by spontaneous breathing or intra-tracheal LPS followed by four hours mechanical ventilation with tidal volumes of 10 mL/kg, respiratory rate of 150 breaths per minute, inspired oxygen 0.21 and positive end expiratory pressure (PEEP) of 3 cm of water. Results Compared with the B6 mice, the lpr mice showed attenuation of the neutrophilic response as measured by decreased numbers of BAL neutrophils and lung myeloperoxidase activity. Interestingly, the B6 and lpr mice had similar concentrations of pro-inflammatory cytokines, including CXCL1 (KC), and similar measurements of permeability and apoptosis. However, the B6 mice showed greater deposition of anti-KC:KC immune complexes in the lungs, as compared with the lpr mice. Conclusions We conclude that a functioning Fas/FasL system is required for full neutrophilic response to LPS in mechanically ventilated mice.

Abstract Background Tetracyclines are broad-spectrum antibiotics that are also used to induce gene expression using the reverse tetracycline transactivator / tetracycline operator system (rtTA/tetO system). The system assumes that tetracyclines have no effects on mammals. However, a number of studies suggest that tetracyclines may have powerful anti-inflammatory effects. We report that the tetracycline, doxycycline, inhibits neutrophil (PMN) influx into the lungs of mice treated with bacterial endotoxin (LPS). Methods Mice were challenged with intratracheal LPS in the presence or absence of doxycyline. bronchoalveolar lavage cell counts and differential, total bronchoalveolar lavage protein, lung homogenate caspase-3 and tissue imaging were used to assess lung injury. In addition, PMN chemotaxis was measured in vitro and syndecan-1 was measured in bronchoalveolar lavage fluid. Results The administration of doxycycline resulted in a significant decrease in the number of bronchoalveolar lavage PMNs in LPS-treated mice. Doxycycline had no effect on other markers of lung injury such as total bronchoalveolar lavage protein and whole lung caspase-3 activity. However, doxycycline resulted in a decrease in shed syndecan-1 in bronchoalveolar lavage fluid. Conclusion We conclude that doxycycline has an important anti-inflammatory effect that can potentially confound the experiments in which the rtTA/tetO system is being used to study the immune response.

This study investigated whether recombinant human soluble Fas ligand (rh-sFasL) induces apoptosis of primary type II pneumocytes in vitro and lung injury in vivo. Type II cells isolated from normal rabbit lung expressed Fas on their surface and became apoptotic after an 18-h incubation with rh-sFasL. Fas expression in normal rabbit lungs was localized by immunohistochemistry to alveolar and airway epithelia and alveolar macrophages. The administration of 10 [micro]g of rh-sFasL into the right lungs of rabbits resulted 24 h later in both significantly more bronchoalveolar lavage fluid total protein and significantly more tissue changes compared with those in the left lungs, which received rh-sFasL plus Fas:Ig (a fusion protein that binds and blocks sFasL). Tissue changes included thickening of the alveolar walls, neutrophilic infiltrates, apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive) cells in the alveolar walls, and increased expression of interleukin-8 by alveolar macrophages (as determined by immunohistochemistry). We conclude that the alveolar epithelium of normal rabbits expresses Fas and that sFasL induces lung injury and inflammation in rabbits. pneumocytes; neutrophils; interleukin-8; macrophages