Metagenomic sequencing and evaluation of the host response in the pediatric aerodigestive population.

OBJECTIVES: To assess the diagnostic utility of metagenomic sequencing in pediatric aerodigestive clinic patients being evaluated for chronic aspiration. We hypothesize that using a metagenomics platform will aid in the identification of microbes not found on standard culture. STUDY DESIGN AND METHODS: Twenty-four children referred to an aerodigestive clinic were enrolled in a prospective, single-site, cross-sectional cohort study. At the time of clinical evaluation under anesthesia, two samples were obtained: an upper airway sample and a sample from bronchoalveolar lavage (BAL). Samples were sent for routine culture and analyzed using Explify® Respiratory, a CLIA Laboratory Developed Test which identifies respiratory commensals and pathogens through RNA and DNA sequencing. Since RNA was sequenced in the course of the metagenomic analysis to identify organisms (RNA viruses and bacteria), the sequencing approach also captured host derived messenger RNA during sample analysis. This incidentally obtained host transcriptomic data were analyzed to evaluate the host immune response. The results of these studies were correlated with the clinical presentation of the research subjects. RESULTS: In 10 patients, organisms primarily associated with oral flora were identified in the BAL. Standard culture was negative in three patients where clinical metagenomics led to a result with potential clinical significance. Transcriptomic data correlated with the presence or absence of dysphagia as identified on prior videofluoroscopic evaluation of swallowing. CONCLUSIONS: Clinical metagenomics allows for simultaneous analysis of the microbiota and the host immune response from BAL samples. As the technologies in this field continue to advance, such testing may improve the diagnostic evaluation of patients with suspected chronic aspiration.

Lentil aspiration leading to likely hypersensitivity pneumonitis.

RATIONALE: A variety of inhaled antigens have been implicated to cause hypersensitivity pneumonitis (HP). We observed that children force-fed with lentil-based weaning food had persistent respiratory symptoms and radiology similar to HP. OBJECTIVES: To describe the clinical features of lentil HP. METHODS: We conducted a retrospective review of records of children with lentil HP attending Pediatric Chest Clinic at a tertiary care hospital in North India from 2008-2018. We included case records with elevated immunoglobulin G (IgG) specific for lentil antigen. MEASUREMENTS AND MAIN RESULTS: Nine children (seven boys) were identified with median (IQR) age of onset of symptoms and diagnosis at 9 (6, 12) and 11 (10, 16) months, respectively. Chronic cough (100%), shortness of breath (89%), fever (78%), vomiting (56%), and wheezing (33%) were common symptoms. Fine crackles were heard in 33% of children, none had clubbing. CT scans showed nodular opacities and consolidation in 78% and 67% children, respectively. Bronchoalveolar lavage showed increased neutrophils and lymphocytes (67% and 33%, respectively). All children showed rapid remission with systemic steroids (prednisolone), starting at a median dose of 1 (1, 1.1) mg kg-1 day-1 . One child had a clinical relapse which was treated with oral steroids again. IgG specific to lentil antigens was elevated in children with lentil HP (21->200 mgA/L) compared with children with other chronic respiratory illnesses (n = 7, <2-11.4 mgA/L). CONCLUSIONS: Lentil aspiration is an important cause of HP in infants of weaning age with force-feeding practices. Further studies are needed to identify aspirated antigens which cause HP in aspiration prone children.

The bacterial microbiota in inflammatory lung diseases.

Numerous lines of evidence, ranging from recent studies back to those in the 1920s, have demonstrated that the lungs are NOT bacteria-free during health. We have recently proposed that the entire respiratory tract should be considered a single ecosystem extending from the nasal and oral cavities to the alveoli, which includes gradients and niches that modulate microbiome dispersion, retention, survival and proliferation. Bacterial exposure and colonization of the lungs during health is most likely constant and transient, respectively. Host microanatomy, cell biology and innate defenses are altered during chronic lung disease, which in turn, alters the dynamics of bacterial turnover in the lungs and can lead to longer term bacterial colonization, as well as blooms of well-recognized respiratory bacterial pathogens. A few new respiratory colonizers have been identified by culture-independent methods, such as Pseudomonas fluorescens; however, the role of these bacteria in respiratory disease remains to be determined.

Metal rich particulate matter impairs acetylcholine-mediated vasorelaxation of microvessels in mice.

BACKGROUND: Exposure to PM2.5 (particulate matter<2.5 µm) has been associated with changes in endothelial function. PM2.5 was collected from two Chinese cities, Jinchang (JC) and Zhangye (ZH), both with similar PM2.5 concentrations. However, JC had levels of nickel (Ni), selenium (Se), copper (Cu), and arsenic (As) that were 76, 25, 17, and 7 fold higher than that measured in ZH, respectively. We used this unique PM sample to delineate the chemical components that drive pulmonary and systemic effects and explore the mechanism(s) by which vascular dysfunction is caused. METHODS: Male FVB/N mice received oropharyngeal aspiration of water or PM2.5 from JC, ZH or ZH spiked with one of the following elements at the same concentrations found in the JC PM (Ni=4.76; As=2.36; Se=0.24; Cu=2.43 µg/mg) followed by evaluation of markers of pulmonary and systemic inflammation. Mesenteric arteries were isolated for gene expression or functional response to various agonists (Phenylephrine, Acetylcholine, and Sodium Nitroprusside) and inhibitors (L-NAME, Apocynin, and VAS2870) ex vivo. RESULTS: Protein and total cell counts from lung lavage revealed significant pulmonary inflammation from ZH (p<0.01) and JC and ZH+NiSO4 (p<0.001) as compared to control and a significant decrease in mesenteric artery relaxation (p<0.001) and this decrease is blunted in the presence of NADPH oxidase inhibitors. Significant increases in gene expression (TNF-α, IL-6, Nos3; p<0.01; NOX4; p<0.05) were observed in JC and ZH+NiSO4, as well as significantly higher concentrations of VEGF and IL-10 (p<0.01, p<0.001; respectively). CONCLUSIONS: Our results indicate that the specific toxicity observed in PM from JC is likely due to the nickel component in the PM. Further, since VAS2870 was the most successful inhibitor to return vessels to baseline relaxation values, NADPH Oxidase is implicated as the primary source of PM-induced O2•-.

Aspiration, localized pulmonary inflammation, and predictors of early-onset bronchiolitis obliterans syndrome after lung transplantation.

BACKGROUND: We hypothesized that immune mediator concentrations in the bronchoalveolar fluid (BALF) are predictive of bronchiolitis obliterans syndrome (BOS) and demonstrate specific patterns of dysregulation, depending on the presence of acute cellular rejection, BOS, aspiration, and timing of lung transplantation. STUDY DESIGN: We prospectively collected 257 BALF samples from 105 lung transplant recipients. The BALF samples were assessed for absolute and differential white blood cell counts and 34 proteins implicated in pulmonary immunity, inflammation, fibrosis, and aspiration. RESULTS: There were elevated BALF concentrations of interleukin (IL)-15, IL-17, basic fibroblast growth factor, tumor necrosis factor-α, and myeloperoxidase, and reduced concentrations of α1-antitrypsin, which were predictive of early-onset BOS. Patients with BOS had an increased percentage of BALF lymphocytes and neutrophils, with a reduced percentage of macrophages (p < 0.05). The BALF concentrations of IL-1ß; IL-8; interferon-γ-induced protein 10; regulated upon activation, normal T-cell expressed and secreted; neutrophil elastase; and pepsin were higher in patients with BOS (p < 0.05). Among those with BOS, BALF concentrations of IL-1RA; IL-8; eotaxin; interferon-γ-induced protein 10; regulated upon activation, normal T-cell expressed and secreted; myeloperoxidase; and neutrophil elastase were positively correlated with time since transplantation (p < 0.01). Those with worse grades of acute cellular rejection had an increased percentage of lymphocytes in their BALF (p < 0.0001) and reduced BALF concentrations of IL-1ß, IL-7, IL-9, IL-12, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-γ, and vascular endothelial growth factor (p ≤ 0.001). Patients with aspiration based on detectable pepsin had increased percentage of neutrophils (p < 0.001) and reduced BALF concentrations of IL-12 (p < 0.001). CONCLUSIONS: The BALF levels of IL-15, IL-17, basic fibroblast growth factor, tumor necrosis factor-α, myeloperoxidase, and α1-antitrypsin at 6 to 12 months after lung transplantation are predictive of early-onset BOS, and those with BOS and aspiration have an augmented chemotactic and inflammatory balance of pulmonary leukocytes and immune mediators. These data justify the surgical prevention of aspiration and argue for the refinement of antirejection regimens.