Src kinase inhibition with dasatinib impairs neutrophil function and clearance of Escherichia coli infection in a murine model of acute lung injury.

BACKGROUND: Neutrophils rapidly respond to and clear infection from tissues, but can also induce tissue damage through excessive degranulation, when acute inflammation proceeds unchecked. A number of key neutrophil functions, including adhesion-dependent degranulation, are controlled by src family kinases. Dasatinib is a potent src inhibitor used in treating patients with chronic myeloid leukaemia and treatment-resistant acute lymphoblastic leukaemia. We hypothesized that dasatinib would attenuate acute inflammation by inhibiting neutrophil recruitment, degranulation and endothelial cell injury, without impairing bacterial clearance, in a murine model of bacteria-induced acute lung injury. C57BL/6 mice received intratracheal Escherichia coli, and were treated with intraperitoneal dasatinib or control. Bacterial clearance, lung injury, and markers of neutrophil recruitment and degranulation were measured. Separately, human blood neutrophils were exposed to dasatinib or control, and the effects on a range of neutrophil functions assessed. RESULTS: Dasatinib was associated with a dose-dependent significant increase in E. coli in the mouse lung, accompanied by impairment of organ function, reflected in significantly increased protein leak across the alveolar-capillary membrane. However, the number of neutrophils entering the lung was unaffected, suggesting that dasatinib impairs neutrophil function independent of migration. Dasatinib did not cause direct toxicity to human neutrophils, but led to significant reductions in phagocytosis of E. coli, adhesion, chemotaxis, generation of superoxide anion and degranulation of primary and secondary granules. However, no biologically important effect of dasatinib on neutrophil degranulation was observed in mice. CONCLUSIONS: Contrary to our starting hypothesis, src kinase inhibition with dasatinib had a detrimental effect on bacterial clearance in the mouse lung and therefore does not represent an attractive therapeutic strategy to treat primary infective lung inflammation. Data from human neutrophils suggest that dasatanib has inhibitory effects on a range of neutrophil functions.

Randomised controlled trial of GM-CSF in critically ill patients with impaired neutrophil phagocytosis.

BACKGROUND: Critically ill patients with impaired neutrophil phagocytosis have significantly increased risk of nosocomial infection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) improves phagocytosis by neutrophils ex vivo. This study tested the hypothesis that GM-CSF improves neutrophil phagocytosis in critically ill patients in whom phagocytosis is known to be impaired. METHODS: This was a multicentre, phase IIa randomised, placebo-controlled clinical trial. Using a personalised medicine approach, only critically ill patients with impaired neutrophil phagocytosis were included. Patients were randomised 1:1 to subcutaneous GM-CSF (3 µg/kg/day) or placebo, once daily for 4 days. The primary outcome measure was neutrophil phagocytosis 2 days after initiation of GM-CSF. Secondary outcomes included neutrophil phagocytosis over time, neutrophil functions other than phagocytosis, monocyte HLA-DR expression and safety. RESULTS: Thirty-eight patients were recruited from five intensive care units (17 randomised to GM-CSF). Mean neutrophil phagocytosis at day 2 was 57.2% (SD 13.2%) in the GM-CSF group and 49.8% (13.4%) in the placebo group, p=0.73. The proportion of patients with neutrophil phagocytosis≥50% at day 2, and monocyte HLA-DR, appeared significantly higher in the GM-CSF group. Neutrophil functions other than phagocytosis did not appear significantly different between the groups. The most common adverse event associated with GM-CSF was fever. CONCLUSIONS: GM-CSF did not improve mean neutrophil phagocytosis at day 2, but was safe and appeared to increase the proportion of patients with adequate phagocytosis. The study suggests proof of principle for a pharmacological effect on neutrophil function in a subset of critically ill patients.

Exchange protein directly activated by cyclic AMP (EPAC) activation reverses neutrophil dysfunction induced by ß2-agonists, corticosteroids, and critical illness.

BACKGROUND: Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired infection. Despite this, remarkably few neutrophil-specific treatments exist. OBJECTIVES: We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. METHODS: Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using ß2-agonists. Inhibitors and activators of cyclic AMP (cAMP)-dependent pathways were used to assess the influence on neutrophil phagocytosis in vitro. RESULTS: ß2-Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by ß2-agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A (PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by ß2-agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. CONCLUSIONS: EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis.

Non cystic fibrosis bronchiectasis: A longitudinal retrospective observational cohort study of Pseudomonas persistence and resistance.

BACKGROUND: The hallmark of non-cystic fibrosis bronchiectasis is recurrent bronchial infection, yet there are significant gaps in our understanding of pathogen persistence, resistance and exacerbation frequencies. Pseudomonas aeruginosa is a key pathogen thought to be a marker of disease severity and progression, yet little is known if the infection risk is seen in those with milder disease or if there is any potential for eradication. These data are important in determining risk stratification and follow up. METHODS AND PATIENT COHORT: A retrospective review of consecutive adult patients attending a specialist UK bronchiectasis clinic over a two-year recruitment period between July 2007 and June 2009 was performed. Analysis of our primary outcome, longitudinal microbiological status, was recorded based on routine clinical follow-up through to data capture point or date of death. Patients were stratified by lung function and infecting organism. RESULTS: 155 patients (mean (SD) age 62.2 (12.4) years; 60.1% female) were identified from clinic records with microbiological data for a median (IQR) follow up duration of 46 (35-62) months. Baseline mean FEV1% predicted was 60.6% (24.8) with mean exacerbation frequency of 4.42/year; 73.6% reported 3 or more exacerbations/year. Haemophilus influenzae was isolated in 90 (58.1%) patients and P. aeruginosa in 78 (50.3%) patients with persistent infection in 51 (56.7%) H. influenzae and 47 (60.3%) P. aeruginosa, respectively. Of the P. aeruginosa colonised patients, 16 (34%) became culture negative on follow-up with a mean of 5.2 negative sputum cultures/patient. P. aeruginosa was isolated from 5 out of 39 patients (12.8%) with minimal airflow limitation as compared to 18 out of 38 patients (47.4%) with severe airflow limitation. Although hospital admissions were significantly higher in the P. aeruginosa infected group (1.3 vs. 0.7 admissions per annum, p = 0.035), overall exacerbation rates were the same (4.6 vs. 4.3, p = 0.58). Independent predictors of P. aeruginosa colonisation were low FEV1% predicted (OR 2.46; 95% CI 1.27-4.77) and polymicrobial colonisation (OR 4.07; 95% CI 1.56-10.58). 17 (11%) patients were infected with multi-resistant strains; however, none were pan-resistant. CONCLUSIONS: P. aeruginosa is associated with greater persistent infection rates and more hospital admissions than H. influenzae. Exacerbation rates, however, were similar; therefore H. influenzae causes significant out-patient morbidity. P. aeruginosa infection occurs across all strata of lung function impairment but is infrequently multi-resistant in bronchiectasis. Careful microbiology follow up is required even in those with well-preserved lung function.

Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia.

BACKGROUND: Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare. OBJECTIVES: We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. METHODS: A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >10(4) colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1ß), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. RESULTS: Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1ß was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1ß and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). CONCLUSIONS: Low BALF IL-1ß in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship.

Effect of granulocyte-macrophage colony-stimulating factor on neutrophil function in idiopathic bronchiectasis.

BACKGROUND AND OBJECTIVE: Neutrophils are consistently found in inflamed and infected airways in idiopathic bronchiectasis, but relatively little is known about the function of blood neutrophils in this condition. We hypothesized that peripheral blood neutrophil (PBN) phagocytosis and superoxide generation are impaired in bronchiectasis, and that granulocyte-macrophage colony-stimulating factor (GM-CSF) is capable of improving neutrophil function. METHODS: Neutrophils were isolated from the peripheral blood of patients with idiopathic bronchiectasis who were free of exacerbation, and from healthy controls of similar age (n = 21 in both groups). Ingestion of serum-opsonized zymosan by neutrophils was used to quantify phagocytic capacity. Superoxide generation in neutrophils was measured in response to addition of platelet activating factor and formyl-methionyl-leucyl-phenylalanine. Experiments were performed in the presence or absence of GM-CSF. RESULTS: No differences were observed in either phagocytic capacity (P = 0.99) or superoxide generation (P = 0.81) when comparing patients and controls. However, a significant increase in phagocytic capacity above baseline levels in both patients (P < 0.005) and controls (P < 0.005) was induced by GM-CSF. Similarly, the superoxide generation in patients (P < 0.005) and controls (P = 0.001) was significantly increased by GM-CSF. CONCLUSIONS: PBN function was preserved in idiopathic bronchiectasis. Enhancement of neutrophil phagocytosis and superoxide generation by GM-CSF requires further study.

Low serum mannose-binding lectin level is not associated with disease severity in non-cystic fibrosis bronchiectasis.

Deficiency of mannose-binding lectin (MBL), a serum protein involved in killing and promoting phagocytosis of pathogens, is associated with respiratory infection and disease progression in a number of acute and chronic lung diseases, including cystic fibrosis (CF)- associated bronchiectasis. No such association has been studied in non-CF bronchiectasis (nCF-Br). One hundred and thirty-three adult patients with nCF-Br were studied. Serum MBL levels were measured and deficiency defined using two cut-off levels, i.e. MBL ≤100 ng/ml and ≤600 ng/ml. Parameters of severity included lung function impairment, annual exacerbation and hospital admission rates, breathlessness, and Pseudomonas aeruginosa and Haemophilus influenzae infection rates. The incidence of MBL deficiency using cut-off levels of 100 ng/ml and 600 ng/ml was 10% and 26% respectively, similar to rates seen in the general population. There was no significant difference in mean FEV(1)% predicted between MBL deficient and sufficient patients at both cut-off levels (≤100 ng/ml: 63.8% vs. 64.6%, P = 0.91; ≤ 600 ng/ml: 66.5% vs. 63.9%, P = 0.56). In addition, exacerbation/hospital admission rates, symptoms of breathlessness and isolation/colonisation rates with P. aeruginosa and H. influenzae were similar in both groups at both cut-off levels. In conclusion, MBL deficiency is not associated with markers of disease severity in patients with nCF-Br.

Stochastic processes in the causation of rheumatic disease.

OBJECTIVE: Rheumatic disorders arise in certain individuals depending on the interaction of genetic and environmental factors, the contribution for each varying with the specific rheumatic disorder. However, a third variable, i.e., random or stochastic processes, may be important, but this has been poorly studied. We examined 3 rheumatic disorders to determine whether a simple stochastic process might be consistent with the incidence data. METHODS: A questionnaire and clinical survey of patients with ankylosing spondylitis, rheumatoid arthritis, and systemic sclerosis was performed to determine age at onset of first symptom. Population data were obtained from the Australian Bureau of Statistics. Computer modeling of the equation dN/dt = kP0 tr-1exp(-ktr/r) was performed, where dN/dt is the age-specific incidence rate, P0 is the proportion of population at risk, t is the age at onset, k is a constant, and r is the number of random events that must occur before the disease manifests. RESULTS: Analysis of the age-specific incidence for each of these 3 rheumatic disorders was consistent with the stochastic model, where r varied from 4 to 9. CONCLUSION: An examination of the age-specific incidence suggests that only a small number of random events need to occur in a predisposed population to allow the emergence of the rheumatic disorder. These random events might be environmental (e.g., infections or exposure to toxins) or due to acquired genetic changes (e.g., somatic mutations involving pivotal immune or growth/repair genes).