GAT107-mediated α7 nicotinic acetylcholine receptor signaling attenuates inflammatory lung injury and mortality in a mouse model of ventilator-associated pneumonia by alleviating macrophage mitochondrial oxidative stress via reducing MnSOD-S-glutathionylation.

Supraphysiological concentrations of oxygen (hyperoxia) can compromise host defense and increase susceptibility to bacterial and viral infections, causing ventilator-associated pneumonia (VAP). Compromised host defense and inflammatory lung injury are mediated, in part, by high extracellular concentrations of HMGB1, which can be decreased by GTS-21, a partial agonist of α7 nicotinic acetylcholine receptor (α7nAChR). Here, we report that a novel α7nAChR agonistic positive allosteric modulator (ago-PAM), GAT107, at 3.3 mg/kg, i.p., significantly decreased animal mortality and markers of inflammatory injury in mice exposed to hyperoxia and subsequently infected with Pseudomonas aeruginosa. The incubation of macrophages with 3.3 µM of GAT107 significantly decreased hyperoxia-induced extracellular HMGB1 accumulation and HMGB1-induced macrophage phagocytic dysfunction. Hyperoxia-compromised macrophage function was correlated with impaired mitochondrial membrane integrity, increased superoxide levels, and decreased manganese superoxide dismutase (MnSOD) activity. This compromised MnSOD activity is due to a significant increase in its level of glutathionylation. The incubation of hyperoxic macrophages with 3.3 µM of GAT107 significantly decreases the levels of glutathionylated MnSOD, and restores MnSOD activity and mitochondrial membrane integrity. Thus, GAT107 restored hyperoxia-compromised phagocytic functions by decreasing HMGB1 release, most likely via a mitochondrial-directed pathway. Overall, our results suggest that GAT107 may be a potential treatment to decrease acute inflammatory lung injury by increasing host defense in patients with VAP.

The Role of Presepsin Obtained from Tracheal Aspirates in the Diagnosis of Early Onset Pneumonia in Intubated Newborns.

OBJECTIVES: To investigate the role of presepsin obtained from tracheal aspirate of intubated newborns in the diagnosis of early neonatal pneumonia. METHODS: A cross-sectional observational study was performed on 60 intubated newborns during the two-year period. Tracheal aspirate for examination was taken in aseptic conditions in usual toilets, by lavage with 2 ml of 0.9% NaCl in Mucus suction set. On the same day, presepsin (blood) was measured. RESULTS: There were 34 newborns in the examined group (with pneumonia) and 26 in the control group. Patient groups were similar regarding demographic characteristics related to gender and Apgar score. The coefficients of simple linear correlation revealed the statistically significant connection between presepsin (from tracheal aspirate) and birth body weight, presepsin (plasma), maternal infection and pneumonia. Significant differences in the values of presepsin (from tracheal aspirate) (p < 0.001) and birth body weight (p = 0.036) were found. CONCLUSIONS: In intubated newborns, measurements of presepsin obtained from tracheal aspirate suggested that it can be used as a complementary marker in diagnosing early onset neonatal pneumonia.

Serum and alveolar procalcitonin had a weak diagnostic value for ventilator-associated pneumonia in patients with pulmonary infection score ≥ 6.

BACKGROUND: Measuring the serum and alveolar procalcitonin level as inflammatory marker in the diagnosis of ventilator-associated pneumonia (VAP) has been taken into account. In this study, serum and alveolar procalcitonin levels in patients with suspected VAP and patients with confirmed VAP were compared. METHODS: This cross-sectional study was conducted using 50 intubated intensive care unit (ICU) patients, connected to ventilator, from October 2014 to April 2015. 50 patients with clinical pulmonary infection score ≥6 were divided into two groups. Patients whose bronchoalveolar lavage (BAL) has shown the growth of more than 104 CFU/mL were included in confirmed VAP group and other patients were included in suspected VAP group. Serum and alveolar procalcitonin levels were measured and compared between both groups. RESULTS: Mean age of patients was 69.10 ± 42.13 with a range of 16-90 years, out of which 23 patients were male (46%) and 27 patients were female (54%). Moreover, patients' mean clinical pulmonary infection score was reported to be 7.02 ± 1.07. There was a significant relationship between serum and alveolar procalcitonin in suspected patients and patients with an approved form of pneumonia (p = 0.001 and 0.027). Area under the curve for alveolar procalcitonin was 0.683 (sensitivity = 57%; specificity = 80%) and for serum procalcitonin 0.751 (sensitivity = 71%; specificity = 73%) for the diagnosis of VAP. CONCLUSION: According to the results of the present study, we can diagnose ventilator-associated pneumonia earlier and more accurately by measuring procalcitonin level (particularly alveolar type) in intensive care unit patients.

The study of vitamin D administration effect on CRP and Interleukin-6 as prognostic biomarkers of ventilator associated pneumonia.

PURPOSE: In regard with the effect of immune-stimulants in the treatment of infectious diseases, the effect of vitamin D administration on the outcome of patients with Ventilator-Associated Pneumonia (VAP) with a high rate of mortality, was studied. MATERIAL AND METHOD: In this trial, 46 adult patients suffering from VAP and vitamin D deficiency were enrolled. The first group of patients received single intramuscular injection of vitamin D (300000Unit), while the other group were given the placebo. RESULTS: Administration of vitamin D significantly enhanced its levels (P<0.0001) in the treated patients (12.28±8.26) in comparison with placebo group (1.15±1.50). Serum Interleukin-6 levels were significantly reduced in the treated group compared to placebo (P=0.01). Although C-Reactive protein (CRP) levels showed an improving trend in the vitamin D group, no significant difference between groups (P=0.12) was found. Interestingly, the mortality rate of patients that treated with vitamin D (5/24) was significantly lower (p=0.04) than that of the placebo group (11/22). CONCLUSION: Our results indicate that vitamin D administration can significantly reduce the IL-6 as prognostic marker in VAP patients, and must be considered as adjunct option in the treatment of VAP patients.

Add-on effect of Qinbei Qingfei Yiqi granules for ventilator-associated pneumonia in intensive care units: a randomized controlled trial.

OBJECTIVE: To evaluate the add-on effect of Qinbei Qingfei Yiqi (QBQFYQ) granules for ventilator-associated pneumonia (VAP) in non-infectious critically ill patients. METHODS: In this randomized controlled trial, 80 non-infectious critically ill patients undergoing mechanical ventilation in the intensive care unit (ICU) were randomly divided into two groups: those receiving QBQFYQ granules plus usual treatment (experimental group, n = 50) and those receiving only the usual treatment (control group, n = 30). The main outcome measures were VAP rate, time of VAP occurrence, duration of mechanical ventilation, and length of ICU stay. Interleukin-6 (IL-6), C-reactive protein (CRP), and T-lymphocyte (CD4+, CD8+, and CD4+/CD8+ ratio) serum levels were also evaluated before and after treatment. RESULTS: Compared with the control group treatment QBQFYQ administration significantly reduced the duration of mechanical ventilation [(9.58 ± 3.14) vs (12.52 ± 4.33) days] and length of ICU stay [(14.57 ± 3.72) vs (17.82 ± 5.24) days] and delayed VAP occurrence [(4.31 ± 0.86) vs (2.43 ± 0.27) days]. Additionally, CRP and IL-6 serum levels and CD4+/CD8+ ratio were significantly lower in the experimental group (P < 0.05) than in the control group. However, there were no significant differences in hospital mortality rate (30.0% vs 33.3% ) and adverse events (4.0% vs 6.7%). adverse events (4.0% vs 6.7%). CONCLUSION: QBQFYQ delays the time of VAP occurrence and shortens the duration of mechanical ventilation in non-infectious critically ill patients, possibly through anti-inflammatory and immunomodulatory mechanisms.

Pseudomonas aeruginosa colonization enhances ventilator-associated pneumonia-induced lung injury.

BACKGROUND: Pseudomonas aeruginosa (PA) is the single-most common pathogen of ventilator-associated pneumonia (VAP). Large quantities of PA in the trachea of ventilated patients are associated with an increased risk of death. However, the role of PA colonization in PA VAP-induced lung injury remains elusive. This study examined the effect and mechanism of PA colonization in VAP-induced lung injury. METHODS: C57BL/6 wild-type (WT) and c-Jun N-terminal kinase knockout (JNK1(-/-)) mice received mechanical ventilation for 3 h at 2 days after receiving nasal instillation of PA (1 × 10(6) colony forming unit) or normal saline. RESULTS: Intranasal instillation of PA or mechanical ventilation induced the expression of interleukin-6 (IL-6) in the lungs. Phospho-JNK protein expression in the lungs was significantly increased in mice receiving mechanical ventilation after PA instillation as compared with those receiving ventilation alone. Mechanical ventilation after PA instillation significantly increased the expression of tumor necrosis factor-α (TNF-α), IL-1ß, and macrophage inflammatory protein-2 (MIP-2) proteins; neutrophil sequestration; and TNF-α, IL-1ß, and IL-6 levels in the lungs of WT mice, but not in JNK1(-/-) mice. CONCLUSION: PA colonization plays an important role in PA VAP-induced lung injury through the induction of JNK1-mediated inflammation. PA-induced VAP causes lung injury through JNK signaling pathway in the lungs. JNK inhibition in ICU patients with higher percentages of PA colonization may reduce VAP-induced lung injury and mortality.

Spinal cord injury modulates the lung inflammatory response in mechanically ventilated rats: a comparative animal study.

Mechanical ventilation (MV) is widely used in spinal injury patients to compensate for respiratory muscle failure. MV is known to induce lung inflammation, while spinal cord injury (SCI) is known to contribute to local inflammatory response. Interaction between MV and SCI was evaluated in order to assess the impact it may have on the pulmonary inflammatory profile. Sprague Dawley rats were anesthetized for 24 h and randomized to receive either MV or not. The MV group included C4-C5 SCI, T10 SCI and uninjured animals. The nonventilated (NV) group included T10 SCI and uninjured animals. Inflammatory cytokine profile, inflammation related to the SCI level, and oxidative stress mediators were measured in the bronchoalveolar lavage (BAL). The cytokine profile in BAL of MV animals showed increased levels of TNF-α, IL-1ß, IL-6 and a decrease in IL-10 (P = 0.007) compared to the NV group. SCI did not modify IL-6 and IL-10 levels either in the MV or the NV groups, but cervical injury induced a decrease in IL-1ß levels in MV animals. Cervical injury also reduced MV-induced pulmonary oxidative stress responses by decreasing isoprostane levels while increasing heme oxygenase-1 level. The thoracic SCI in NV animals increased M-CSF expression and promoted antioxidant pulmonary responses with low isoprostane and high heme oxygenase-1 levels. SCI shows a positive impact on MV-induced pulmonary inflammation, modulating specific lung immune and oxidative stress responses. Inflammation induced by MV and SCI interact closely and may have strong clinical implications since effective treatment of ventilated SCI patients may amplify pulmonary biotrauma.

Gene expression profile analysis of ventilator-associated pneumonia.

Based on the gene expression profile of patients with ventilator-associated pneumonia (VAP) and patients not affected by the disease, the present study aimed to enhance the current understanding of VAP development using bioinformatics methods. The expression profile GSE30385 was downloaded from the Gene Expression Omnibus database. The Linear Models for Microarray Data package in R language was used to screen and identify differentially expressed genes (DEGs), which were grouped as up­ and down­regulated genes. The up­ and downregulated genes were functionally enriched using the Database for Annotation, Visualization and Integrated Discovery system and then annotated according to TRANSFAC, Tumor Suppressor Gene and Tumor Associated Gene databases. Subsequently, the protein­protein interaction (PPI) network was constructed, followed by module analysis using CFinder software. A total of 69 DEGs, including 33 up­ and 36 downregulated genes were screened out in patients with VAP. Upregulated genes were mainly enriched in functions and pathways associated with the immune response (including the genes ELANE and LTF) and the mitogen-activated protein kinase (MAPK) signaling pathway (including MAPK14). The PPI network comprised 64 PPI pairs and 44 nodes. The top two modules were enriched in different pathways, including the MAPK signaling pathway. Genes including ELANE, LTF and MAPK14 may have important roles in the development of VAP via altering the immune response and the MAPK signaling pathway.

The development of early-onset ventilator-associated pneumonia after cardiac surgery with cardiopulmonary bypass is associated with toll-like receptor 4 signal transduction pathways.

We evaluated the hypothesis that early-onset ventilator-associated pneumonia (VAP) after cardiac surgery with cardiopulmonary bypass (CPB) requires Toll-like receptor 4 (TLR4)-dependent signaling pathways. We enrolled 50 patients undergoing mitral and aortic double valve replacement, collecting left atrial blood samples from all patients preoperatively (T1), when opening the atrial septum (T2), closing the atrial septum (T3), and at the end of CPB (T4). TLR4 expression on monocytes and lymphocytes was detected immediately with flow cytometry. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-10 (IL-10) were measured using enzyme-linked immunosorbent assays (ELISA). Data from flow cytometry, ELISA, and the clinical outcomes in the two groups were evaluated and compared. Seventeen patients were included for analysis, and assigned to group A (without early-onset VAP, n = 10) and group B (with early-onset VAP, n = 7). TLR4 expression on lymphocytes in group B at T1, T3, and T4 were significantly higher than those in group A. Serum levels of IL-10 in group A at T4 were significantly higher than for group B. We found characteristic patterns in TLR4 expression on lymphocytes in left atrial blood of patients undergoing cardiac surgery with CPB. Our findings clarify the role of TLR4 and its association with the development of postoperative pneumonia.

A biomarker panel (Bioscore) incorporating monocytic surface and soluble TREM-1 has high discriminative value for ventilator-associated pneumonia: a prospective observational study.

INTRODUCTION: Ventilator-associated pneumonia (VAP) increases mortality in critical illness. However, clinical diagnostic uncertainty persists. We hypothesised that measuring cell-surface and soluble inflammatory markers, incorporating Triggering Receptor Expressed by Myeloid cells (TREM)-1, would improve diagnostic accuracy. METHODS: A single centre prospective observational study, set in a University Hospital medical-surgical intensive Care unit, recruited 91 patients into 3 groups: 27 patients with VAP, 33 ventilated controls without evidence of pulmonary sepsis (non-VAP), and 31 non-ventilated controls (NVC), without clinical infection, attending for bronchoscopy. Paired samples of Bronchiolo-alveolar lavage fluid (BALF) and blood from each subject were analysed for putative biomarkers of infection: Cellular (TREM-1, CD11b and CD62L) and soluble (IL-1ß, IL-6, IL-8, sTREM-1, Procalcitonin). Expression of cellular markers on monocytes and neutrophils were measured by flow cytometry. Soluble inflammatory markers were determined by ELISA. A biomarker panel ('Bioscore'), was constructed, tested and validated, using Fisher's discriminant function analysis, to assess its value in distinguishing VAP from non VAP. RESULTS: The expression of TREM-1 on monocytes (mTREM-1) and neutrophils (nTREM-1) and concentrations of IL-1ß, IL-8, and sTREM-1 in BALF were significantly higher in VAP compared with non-VAP and NVC (p<0.001). The BALF/blood mTREM-1 was significantly higher in VAP patients compared to non-VAP and NVC (0.8 v 0.4 v 0.3 p<0.001). A seven marker Bioscore (BALF/blood ratio mTREM-1 and mCD11b, BALF sTREM-1, IL-8 and IL-1ß, and serum CRP and IL-6) correctly identified 88.9% of VAP cases and 100% of non-VAP cases. CONCLUSION: A 7-marker bioscore, incorporating cellular and soluble TREM-1, accurately discriminates VAP from non-pulmonary infection.

The α7 nicotinic acetylcholine receptor agonist GTS-21 improves bacterial clearance in mice by restoring hyperoxia-compromised macrophage function.

Mechanical ventilation with supraphysiological concentrations of oxygen (hyperoxia) is routinely used to treat patients with respiratory distress. However, prolonged exposure to hyperoxia compromises the ability of the macrophage to phagocytose and clear bacteria. Previously, we showed that the exposure of mice to hyperoxia elicits the release of the nuclear protein high mobility group box-1 (HMGB1) into the airways. Extracellular HMGB1 impairs macrophage phagocytosis and increases the mortality of mice infected with Pseudomonas aeruginosa (PA). The aim of this study was to determine whether GTS-21 [3-(2,4 dimethoxybenzylidene)-anabaseine dihydrochloride], an α7 nicotinic acetylcholine receptor (α7nAChR) agonist, could inhibit hyperoxia-induced HMGB1 release into the airways, enhance macrophage function and improve bacterial clearance from the lungs in a mouse model of ventilator-associated pneumonia. GTS-21 (0.04, 0.4 and 4 mg/kg) or saline was systemically administered via intraperitoneal injection to mice that were exposed to hyperoxia (≥99% O2) and subsequently challenged with PA. We found that systemic administration of 4 mg/kg GTS-21 significantly increased bacterial clearance, decreased acute lung injury and decreased accumulation of airway HMGB1. To investigate the cellular mechanism of these observations, RAW 264.7 cells, a macrophagelike cell line, were incubated with different concentrations of GTS-21 in the presence of 95% O2. The phagocytic activity of macrophages was significantly increased by GTS-21 in a dose-dependent manner. In addition, hyperoxia-induced hyperacetylation of HMGB1 was significantly reduced in macrophages incubated with GTS-21. Furthermore, GTS-21 significantly inhibited the cytoplasmic translocation and release of HMGB1 from these macrophages. Our results indicate that GTS-21 is effective in improving bacterial clearance and reducing acute lung injury by enhancing macrophage function via inhibiting the release of nuclear HMGB1. Therefore, the α7nAChR represents a possible pharmacological target to improve the clinical outcome of patients on ventilators by augmenting host defense against bacterial infections.

Midkine is expressed and differentially processed during chronic obstructive pulmonary disease exacerbations and ventilator-associated pneumonia associated with Staphylococcus aureus infection.

Staphylococcus aureus is sometimes isolated from the airways during acute exacerbations of chronic obstructive pulmonary disease (COPD) but more commonly recognized as a cause of ventilator-associated pneumonia (VAP). Antimicrobial proteins, among them midkine (MK), are an important part of innate immunity in the airways. In this study, the levels and possible processing of MK in relation to S. aureus infection of the airways were investigated, comparing COPD and VAP, thus comparing a state of disease with preceding chronic inflammation and remodeling (COPD) with acute inflammation (that is, VAP). MK was detected in the small airways and alveoli of COPD lung tissue but less so in normal lung tissue. MK at below micromolar concentrations killed S. aureus in vitro. Proteolytic processing of MK by the staphylococcal metalloprotease aureolysin (AL), but not cysteine protease staphopain A (SA), resulted in impaired bactericidal activity. Degradation was seen foremost in the COOH-terminal portion of the molecule that harbors high bactericidal activity. In addition, MK was detected in sputum from patients suffering from VAP caused by S. aureus but less so in sputum from COPD exacerbations associated with the same bacterium. Recombinant MK was degraded more rapidly in sputum from the COPD patients than from the VAP patients and a greater proteolytic activity in COPD sputum was confirmed by zymography. Taken together, proteases of both bacteria and the host contribute to degradation of the antibacterial protein MK, resulting in an impaired defense of the airways, in particular, in COPD where the state of chronic inflammation could be of importance.

High Mobility Group Box-1 mediates hyperoxia-induced impairment of Pseudomonas aeruginosa clearance and inflammatory lung injury in mice.

Mechanical ventilation with supraphysiological concentrations of oxygen (hyperoxia) is routinely used to treat patients with respiratory distress. However, a significant number of patients on ventilators exhibit enhanced susceptibility to infections and develop ventilator-associated pneumonia (VAP). Pseudomonas aeruginosa (PA) is one of the most common species of bacteria found in these patients. Previously, we demonstrated that prolonged exposure to hyperoxia can compromise the ability of alveolar macrophages (AMs), an essential part of the innate immunity, to phagocytose PA. This study sought to investigate the potential molecular mechanisms underlying hyperoxia-compromised innate immunity against bacterial infection in a murine model of PA pneumonia. Here, we show that exposure to hyperoxia (≥ 99% O2) led to a significant elevation in concentrations of airway high mobility group box-1 (HMGB1) and increased mortality in C57BL/6 mice infected with PA. Treatment of these mice with a neutralizing anti-HMGB1 monoclonal antibody (mAb) resulted in a reduction in bacterial counts, injury, and numbers of neutrophils in the lungs, and an increase in leukocyte phagocytic activity compared with mice receiving control mAb. This improved phagocytic function was associated with reduced concentrations of airway HMGB1. The correlation between phagocytic activity and concentrations of extracellular HMGB1 was also observed in cultured macrophages. These results indicate a pathogenic role for HMGB1 in hyperoxia-induced impairment with regard to a host's ability to clear bacteria and inflammatory lung injury. Thus, HMGB1 may provide a novel molecular target for improving hyperoxia-compromised innate immunity in patients with VAP.