Lentiviral delivery of RNAi for in vivo lineage-specific modulation of gene expression in mouse lung macrophages.

Although RNA interference (RNAi) has become a ubiquitous laboratory tool since its discovery 12 years ago, in vivo delivery to selected cell types remains a major technical challenge. Here, we report the use of lentiviral vectors for long-term in vivo delivery of RNAi selectively to resident alveolar macrophages (AMs), key immune effector cells in the lung. We demonstrate the therapeutic potential of this approach by RNAi-based downregulation of p65 (RelA), a component of the pro-inflammatory transcriptional regulator, nuclear factor κB (NF-κB) and a key participant in lung disease pathogenesis. In vivo RNAi delivery results in decreased induction of NF-κB and downstream neutrophilic chemokines in transduced AMs as well as attenuated lung neutrophilia following stimulation with lipopolysaccharide (LPS). Through concurrent delivery of a novel lentiviral reporter vector (lenti-NF-κB-luc-GFP) we track in vivo expression of NF-κB target genes in real time, a critical step towards extending RNAi-based therapy to longstanding lung diseases. Application of this system reveals that resident AMs persist in the airspaces of mice following the resolution of LPS-induced inflammation, thus allowing these localized cells to be used as effective vehicles for prolonged RNAi delivery in disease settings.

Complications, consequences, and practice patterns of endobronchial ultrasound-guided transbronchial needle aspiration: Results of the AQuIRE registry.

BACKGROUND: Few studies of endobronchial ultrasound-guided transbronchial needle aspiration(EBUS-TBNA) have been large enough to identify risk factors for complications. The primary objective of this study was to quantify the incidence of and risk factors for complications in patients undergoing EBUS-TBNA. METHODS: Data on prospectively enrolled patients undergoing EBUS-TBNA in the American College of Chest Physicians Quality Improvement Registry, Evaluation, and Education (AQuIRE)database were extracted and analyzed for the incidence, consequences, and predictors of complications. RESULTS: We enrolled 1,317 patients at six hospitals. Complications occurred in 19 patients (1.44%;95% CI, 0.87%-2.24%). Transbronchial lung biopsy (TBBx) was the only risk factor for complications,which occurred in 3.21% of patients who underwent the procedure and in 1.15% of those who did not (OR, 2.85; 95% CI, 1.07-7.59; P 5 .04). Pneumothorax occurred in seven patients(0.53%; 95% CI, 0.21%-1.09%). Escalations in level of care occurred in 14 patients (1.06%;95% CI, 0.58%-1.78%); its risk factors were age . 70 years (OR, 4.06; 95% CI, 1.36-12.12; P 5 .012),inpatient status (OR, 4.93; 95% CI, 1.30-18.74; P 5 .019), and undergoing deep sedation or general anesthesia (OR, 4.68; 95% CI, 1.02-21.61; P 5 .048). TBBx was performed in only 12.6% of patients when rapid on site cytologic evaluation (ROSE ) was used and in 19.1% when it was not used ( P 5 .006).Interhospital variation in TBBx use when ROSE was used was significant ( P , .001). CONCLUSIONS: TBBx was the only risk factor for complications during EBUS-TBNA procedures.ROSE significantly reduced the use of TBBx.

Pulmonary alveolar proteinosis: a bench-to-bedside story of granulocyte-macrophage colony-stimulating factor dysfunction.

Pulmonary alveolar proteinosis (PAP) is a rare disorder characterized by ineffective clearance of surfactant by alveolar macrophages. Through recent studies with genetically altered mice, the etiology of this idiopathic disease is becoming clearer. Functional deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) appears to contribute to disease pathogenesis because mutant mice deficient in GM-CSF or its receptor spontaneously develop PAP. Recent human studies further suggest a connection between PAP and defective GM-CSF activity because inactivating anti-GM-CSF autoantibodies are observed in all patients with idiopathic PAP, and additional rare cases of PAP in children have been accompanied by genetic defects in the alpha chain of the GM-CSF receptor. In patients and mouse models of PAP, deficient GM-CSF activity appears to result in defective alveolar macrophages that are unable to maintain pulmonary surfactant homeostasis and display defective phagocytic and antigen-presenting capabilities. The most recent studies also suggest that neutrophil dysfunction additionally contributes to the increased susceptibility to lung infections seen in PAP. Because the phenotypic and immunologic abnormalities of PAP in mouse models can be corrected by GM-CSF reconstituting therapies, early clinical trials are underway utilizing administration of GM-CSF to potentially treat human PAP. The development of novel treatment approaches for PAP represents a dramatic illustration in pulmonary medicine of the "bench-to-bedside" process, in which basic scientists, translational researchers, and clinicians have joined together to rapidly take advantage of the unexpected observations frequently made in the modern molecular biology research laboratory.