Faculty Bibliography

Air pollution has been found to cause significant global mortality, with 6.8 million excess deaths attributed to air pollution each year, and similarly large numbers of exacerbations of asthma, chronic obstructive pulmonary disease, and cardiovascular diseases. Epidemiological research has identified associations, and experimental human exposure has provided critical information on dose-response relationships of adverse effects caused by controlled human exposure to individual pollutants. Human exposures further enable examination of the relationship of adverse effects such as symptoms and pulmonary function changes to presumed mechanisms of disease revealed through analysis of bronchoalveolar lavage fluid obtained from the lower respiratory tract. In this Perspective, we analyze the ethics of human exposure, the importance of the information gained, and the risks of such exposure. We find that these studies appear to have been done with proper approval of institutional review boards, were done with informed consent from the participants, and have rarely been associated with serious adverse events.

Each year, millions of pulmonary nodules are discovered by computed tomography and subsequently biopsied. Because most of these nodules are benign, many patients undergo unnecessary and costly invasive procedures. We present a 13-protein blood-based classifier that differentiates malignant and benign nodules with high confidence, thereby providing a diagnostic tool to avoid invasive biopsy on benign nodules. Using a systems biology strategy, we identified 371 protein candidates and developed a multiple reaction monitoring (MRM) assay for each. The MRM assays were applied in a three-site discovery study (n = 143) on plasma samples from patients with benign and stage IA lung cancer matched for nodule size, age, gender, and clinical site, producing a 13-protein classifier. The classifier was validated on an independent set of plasma samples (n = 104), exhibiting a negative predictive value (NPV) of 90%. Validation performance on samples from a nondiscovery clinical site showed an NPV of 94%, indicating the general effectiveness of the classifier. A pathway analysis demonstrated that the classifier proteins are likely modulated by a few transcription regulators (NF2L2, AHR, MYC, and FOS) that are associated with lung cancer, lung inflammation, and oxidative stress networks. The classifier score was independent of patient nodule size, smoking history, and age, which are risk factors used for clinical management of pulmonary nodules. Thus, this molecular test provides a potential complementary tool to help physicians in lung cancer diagnosis.

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe's disease. To investigate whether we could generate a functional recombinant human GAA enzyme (tobrhGAA) in tobacco seeds for future enzyme replacement therapy, we subcloned the human GAA cDNA into the plant expression plasmid-pBI101 under the control of the soybean beta-conglycinin seed-specific promoter and biochemically analyzed the tobrhGAA. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. We found the tobrhGAA to be enzymatically active was readily taken up by GSDII fibroblasts and in white blood cells from whole blood to reverse the defect. The tobrhGAA corrected the enzyme defect in tissues at 7 days after a single dose following intraperitoneal (IP) administration in GAA knockout (GAA-/-) mice. Additionally, we could purify the tobrhGAA since it bound tightly to the matrix of Sephadex G100 and can be eluted by competition with maltose. These data demonstrate indirectly that the tobrhGAA is fully functional, predominantly proteolytically cleaved and contains the minimal phosphorylation and mannose-6-phosphate residues essential for biological activity.

INTRODUCTION:: Lung cancer is the highest cause of mortality among tumor pathologies worldwide. There are no validated techniques for an early detection of pulmonary cancer lesions other than low-dose helical computed tomography scan. Unfortunately, this method has some negative effects. Recent studies have laid the basis for development of exosomes-based techniques to screen/diagnose lung cancers. As the isolation of circulating exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostic applications. METHODS:: We used a first set of 30 plasma samples from as many patients, including 10 patients affected by lung adenocarcinomas, 10 with lung granulomas, and 10 healthy smokers matched for age and sex as negative controls. Wide-range microRNAs analysis (742 microRNAs) was performed by quantitative real time polymerase chain reaction. Data were compared on the basis of lesion characteristics, using WEKA software for statistics and modeling. Subsequently, selected microRNAs were evaluated on an independent larger group of samples (105 specimens: 50 lung adenocarcinomas, 30 lung granulomas, and 25 healthy smokers). RESULTS:: This analysis led to the selection of four microRNAs to perform a screening test (miR-378a, miR-379, miR-139-5p, and miR-200b-5p), useful to divide population into two groups: nodule (lung adenocarcinomas + carcinomas) and non-nodule (healthy former smokers). Six microRNAs (miR-151a-5p, miR-30a-3p, miR-200b-5p, miR-629, miR-100, and miR-154-3p) were selected for a second test on the nodule population to discriminate between lung adenocarcinoma and granuloma. CONCLUSIONS:: The screening test showed 97.5% sensitivity, 72.0% specificity, and area under the curve receiver operating characteristic of 90.8%. The diagnostic test had 96.0% sensitivity, 60.0% specificity, and area under the curve receiver operating characteristic of 76.0%. Further evaluation is needed to confirm the predictive power of these models on larger cohorts of samples.

PURPOSE: World Trade Center (WTC) exposure caused airflow obstruction years after exposure. Chitinases and IgE are innate and humoral mediators of obstructive airway disease. We investigated if serum expression of chitinases and IgE early after WTC exposure predicts subsequent obstruction. METHODS: With a nested case-control design, 251 FDNY personnel had chitotriosidase, YKL-40 and IgE measured in serum drawn within months of 9/11/2001. The main outcome was subsequent Forced Expiratory Volume after 1 second/Forced Vital Capacity (FEV1/FVC) less than the lower limit of normal (LLN). Cases (N = 125) had abnormal FEV1/FVC whereas controls had normal FEV1/FVC (N = 126). In a secondary analysis, resistant cases (N = 66) had FEV1 (>/=107 %) one standard deviation above the mean. Logistic regression adjusted for age, BMI, exposure intensity and post-exposure FEV1/FVC modeled the association between early biomarkers and later lung function. RESULTS: Cases and Controls initially lost lung function. Controls recovered to pre-9/11 FEV1 and FVC while cases continue to decline. Cases expressed lower serum chitotriosidase and higher IgE levels. Increase in IgE increased the odds of airflow obstruction and decreased the odds of above average FEV1. Alternately, increasing chitotriosidase decreased the odds of abnormal FEV1/FVC and increased the odds of FEV1 >/= 107 %. Serum YKL-40 was not associated with FEV1/FVC or FEV1 in this cohort. CONCLUSIONS: Increased serum chitotriosidase reduces the odds of developing obstruction after WTC-particulate matter exposure and is associated with recovery of lung function. Alternately, elevated IgE is a risk factor for airflow obstruction and progressive lung function decline.

BACKGROUND: The lung microbiome of healthy individuals frequently harbors oral organisms. Despite evidence that microaspiration is commonly associated with smoking-related lung diseases, the effects of lung microbiome enrichment with upper airway taxa on inflammation has not been studied. We hypothesize that the presence of oral microorganisms in the lung microbiome is associated with enhanced pulmonary inflammation. To test this, we sampled bronchoalveolar lavage (BAL) from the lower airways of 29 asymptomatic subjects (nine never-smokers, 14 former-smokers, and six current-smokers). We quantified, amplified, and sequenced 16S rRNA genes from BAL samples by qPCR and 454 sequencing. Pulmonary inflammation was assessed by exhaled nitric oxide (eNO), BAL lymphocytes, and neutrophils. RESULTS: BAL had lower total 16S than supraglottic samples and higher than saline background. Bacterial communities in the lower airway clustered in two distinct groups that we designated as pneumotypes. The rRNA gene concentration and microbial community of the first pneumotype was similar to that of the saline background. The second pneumotype had higher rRNA gene concentration and higher relative abundance of supraglottic-characteristic taxa (SCT), such as Veillonella and Prevotella, and we called it pneumotypeSCT. Smoking had no effect on pneumotype allocation, alpha, or beta diversity. PneumotypeSCT was associated with higher BAL lymphocyte-count (P= 0.007), BAL neutrophil-count (P= 0.034), and eNO (P= 0.022). CONCLUSION: A pneumotype with high relative abundance of supraglottic-characteristic taxa is associated with enhanced subclinical lung inflammation.

BACKGROUND: Active TB disease can destroy lung parenchyma leading to cavities. Immune responses that predispose or protect individuals from lung damage during TB are poorly defined. OBJECTIVE: To sample lung immune cells and assay bronchoalveolar lavage (BAL) cell cytokine production. DESIGN: Enrolled subjects (n = 73) had bilateral infiltrates and underwent BAL. RESULTS: All had sputum culture demonstrating Mycobacterium tuberculosis and 22/73 (30%) had cavities on their chest radiograph. Those with cavities at presentation had a higher percentage of polymorphonuclear neutrophils (PMN) in BAL as well as lower inducible protein (IP) 10 (P < 0.01) and interleukin (IL) 6 (P = 0.013) in BAL cell supernatants compared to those without cavities. There was no correlation between cavities and other BAL or serum cytokines. IP-10 was negatively associated with BAL PMN. IP-10 and IL-6 expression above median reduces the odds of cavities by 79% and 78% in logistic regression models. IP-10 and IL-6 clustered with interferon-gamma and tumour necrosis factor-alpha in a principal component analysis, while IL-4 clustered with PMN. CONCLUSION: Increasing IP-10 and IL-6 production by BAL cells is associated with non-cavitary TB in patients who present with radiographically advanced TB. IP-10 and IL-6 may reflect an effective T-helper 1 immune control pathway for TB, attenuating tuberculous lung destruction.

Sonic Hedgehog (Shh) signals from epithelium to mesenchyme during embryonic lung development, but the roles of Hedgehog (Hh) signaling in postnatal lung development and adult lung are not known. Using Gli1nlacZ reporter mice to identify cells with active Hh signaling, we found that Gli1nlacZ-positive mesenchymal cells are densely and diffusely present up to 2 weeks after birth and decline in number thereafter. In adult mice, Gli1nlacZ-positive cells are present around large airways and vessels and are sparse in alveolar septa. Hh-stimulated cells are mostly fibroblasts; only 10% of Gli1nlacZ-positive cells are smooth muscle cells, and most smooth muscle cells do not have activation of Hh signaling. After bleomycin injury there are abundant Gli1nlacZ-positive mesenchymal cells in fibrotic lesions and increased numbers of Gli1nlacZ-positive cells in preserved alveolar septa. Inhibition of Hh signaling with an antibody against all Hedgehog isoforms does not reduce bleomycin-induced fibrosis, but adenovirus-mediated over-expression of Shh increases collagen production in this model. Inhibition of Hh signaling during early postnatal lung development causes airspace enlargement without diminished alveolar septation. Reduction of Hh signaling in the later stages of postnatal lung development may be required for normal thinning and maturation of alveolar septa.

Pulmonary vascular loss is an early feature of chronic obstructive pulmonary disease. Biomarkers of inflammation and of metabolic syndrome, predicts loss of lung function in World Trade Center Lung Injury(WTC-LI). We investigated if other cardiovascular disease (CVD) biomarkers also predicted WTC-LI.This nested case-cohort study used 801 never smoker, WTC exposed firefighters with normal pre-9/11 lung function presenting for subspecialty pulmonary evaluation (SPE) before March, 2008. A representative sub-cohort of 124/801 with serum drawn within six months of 9/11 defined CVD biomarker distribution. Post-9/11/01 FEV1 at subspecialty exam defined cases: susceptible WTC-LI cases with FEV1/=107% (68/801). All models were adjusted for WTC exposure intensity, BMI at SPE, age at 9/11, and pre-9/11 FEV1.Susceptible WTC-LI cases had higher levels of Apo-AII, CRP, and MIP-4 with significant RRs of 3.85, 3.93, and 0.26 respectively with an area under the curve (AUC) of 0.858. Resistant WTC-LI cases had significantly higher sVCAM and lower MPO with RRs of 2.24, and 2.89 respectively; AUC 0.830.Biomarkers of CVD in serum six-month post-9/11 predicted either susceptibility or resistance to WTC-LI. These biomarkers may define pathways producing or protecting subjects from pulmonary vascular disease and associated loss of lung function after an irritant exposure.