Faculty Bibliography

Introduction: Interferon gamma release assays (IGRA) have been widely accepted to replace the tuberculin skin test (TST). Data from the Botswana isoniazid preventative therapy (IPT) study showed that in HIV infected individuals only those who were TST positive benefitted from IPT (92% reduction in TB incidence.) We set out to compare the utility of IGRA's (QuantiFERON Gold-in-tube (QFT-GIT) and TSPOT-TB (TSPOT)) and the TST in a high TB-HIV burden setting. Methods: TST (2TU PPD), QFT-GIT and TSPOT-TB where performed in a prospective cohort of 200 HIV positive individuals on ARV's in Cape Town South Africa. Multivariate analysis was performed to assess associations with clinical, demographic and HIV associated (ARV usage, CD4 count, viral load) factors. Results: The participants had a mean (SD) age of 38.4(8.4) years, were predominantly black African (81.3%), female (53.4%) and 32.9% were ever smokers. The median (range) length of time on ARV's 2.1(0.25-7.56) years with median (range) CD4 count of 380(25-1227). Twenty eight (18.6%) participants were TST positive (>5mm), 45(30%) TSPOT-TB positive and 62(41.3%) QFT positive. The agreement between tests was generally fair/poor: QFT-TSPOT: 72%; kappa 0.39(95%CI 0.23-0.55), QFT-TST: 68%; kappa 0.27(95%CI 0.10- 0.44), TSPOT-TST 67%; kappa 0.13(95%CI -0.08 - 0.33). Using the TST as the gold standard, QFT had a sensitivity of 81.2%, specificity of 65.6% and TSPOT 47.6% & 70.8% respectively. LTBI test status was not different in those with/without a history of previous TB or history of smoking. Conclusions: IGRA's only correctly identified 2/3 of TST positive persons. If used alone, potentially a third of HIV infected individuals will receive unnecessary IPT. Longitudinal follow up studies are required to define the utility of IGRAs in the treatment of LTBI in HIV infected persons

Introduction: Lung cancer is the leading causes of cancer death with an overall survival of 15% at 5 years. The poor survival is primarily because most of the patients present at advanced stage when a surgical resection is no longer feasible. Tobacco smoking is the single most important cause of lung cancer. We hypothesized that sputum would be a specific and sensitive biomarker that is easily accessible and non-invasive to detect lung tumorigenesis at early stage among high-risk smokers. Rationale: Sputum contains upper and lower airway epithelial cells, suggesting the molecular alterations identified in sputum likely reflects tumor-associated changes in the lung. Three genes were chosen to be examined using DNA derived from sputum based on their implication in lung tumorigenesis. STK11 is a serine/threonine kinase that regulates cell polarity and metabolism and functions as a tumor suppressor. Germline mutation of STK11 results in the Peutz-Jeghers syndrome, a familial cancer syndrome characterized an increased risk for epithelial cancers, including non-small cell lung cancer (NSCLC) where STK11 is found to be mutated at 30-40% frequency. CDKN2A (also known as p16) is another tumor suppressor protein that plays an important role in regulating the cell cycle, and mutations in p16 increase the risk of developing a variety of cancers including lung cancer and importantly, its loss of function has been implicated in the early stage of tumorigenesis. p53, a tumor suppressor that is the most frequently mutated gene in lung adenocarcinoma, responds to diverse cellular stresses to regulate target genes that induce cell cycle arrest, apoptosis, senescence, or DNA repair. This DNA-binding protein is postulated to bind to a p53-binding site and activate expression of downstream genes that inhibit growth and/or invasion, which is often impaired by mutations identified in various human cancers. Methods: Spontaneous sputum was collected from normal smokers and lung cancer patients and processed to extract genomic DNA. Primers were designed to span exons 1-2 in CDKN2A, exons 2-9 in TP53 and exons 1-9 in STK11. Amplicons were analyzed by direct sequencing. Results: We identified numerous sequence variations in all three genes examined, including verified polymorphisms and mutations in the introns and exons. Most frequently, we detected G to A substitution in the 5' UTR region in CDKN2A and C to G substitution, which results in P72R in exon 4 in TP53. Conclusion: This panel of genes may be useful biomarkers for early detection of lung cancer among smokers

Rationale: Lung cancer is the leading cause of death from all types of cancer worldwide. The high mortality rate is partly attributed to the lack of methods for early detection and identification of patients who are at risk for developing disease. Since adenocarcinoma in the lung periphery is the most common type of lung cancer and the earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the peripheral airways, we hypothesize that identifying gene expression signatures in the peripheral airways will provide the best approach for better understanding field carcinogenesis of lung adenocarcinomas. To this end, we performed gene and miRNA profiling of cells obtained from brushings of peripheral airways of smokers with and without lung adenocarcinomas. These studies will provide not only mechanistic insights into field carcinogenesis of adenocarcinoma, but more importantly, potential tools for the early detection of lung adenocarcinomas. To this end, we performed gene expression profiling of the peripheral small airway epithelium of patients with lung cancer and compared it to that of non-cancerous smokers to identify gene expression signatures of field cancerization in lung cancer. Methods: Fiberoptic bronchoscopies with brushings were used to collect peripheral airway epithelial cells from the 10^th-12^th-order bronchi in non-cancerous smokers or from the unaffected lobe of suspected lung cancer patients. Clinical samples obtained from the suspected lung cancer patients confirmed the histological diagnosis of primary lung cancer. Using Affymetrix HG-U133A Plus 2.0 microarrays, we performed gene expression profiling of over 47,000 genes using total RNA isolated from small airway epithelial cells obtained from brushings of both lung cancer patients and non-cancerous smokers. Results: Microarray analysis of the gene expression profile in the peripheral airway epithelium demonstrated several up-regulated and down-regulated genes which could represent significant alterations in lung cancer. Significantly up-regulated genes included pro-platelet basic protein, gremlin 1, aminolevulinate synthase 2, sparc/osteonectin, and wnt inhibitory factor 1 while down-regulated genes include cytochrome P450 1B1 and 2A6, mucin 2, aldedyhe dehydrognease 3A1, and glutathione peroxidase 2 (p value < 0.05). Conclusion: We had identified several up-regulated and down-regulated genes in peripheral airway epithelial cells of cancer patients which were significantly different when compared to non-cancerous smokers. Identification of the gene expression signatures in field carcinogenesis will not only provide mechanistic insights into lung cancer, but also allow us to identify novel biomarkers for the early detection of lung cancer

Introduction The lung is a common target of HIV-associated morbidity, but there are few studies of long-term pulmonary consequences of HIV infection after successful introduction of antiretroviral drugs (ARVs), particularly in developing countries with high rates of tuberculosis. Methods: A cross-sectional analysis of a prospective cohort of 152 participants with HIV infection, stable on antiretroviral therapy for at least 3 months, and without features of acute respiratory disease, was performed. Clinical and demographic information was recorded in a respiratory questionnaire, and pulmonary function tests including flow-volume loops before and after bronchodilator, body plethysmography and measurement of diffusing capacity were performed. Results: The participants were predominantly black African (81.3%) and female (53.4%); mean age was 38.4 (SD+/-8.4) years, and 32.9% had a history of tobacco smoking. The median duration of time on ART was 2.1 years (range 0.25-7.56), and the median CD₄ was 380 (range 25-1227). Forty-one percent reported a history of previous tuberculosis, and only 3 subjects previous Pneumocystis pneumonia. None were users of intravenous drugs. Respiratory symptoms were reported in 31% of participants, and any respiratory symptom (cough, phlegm, wheeze or dyspnea) was strongly associated with current smoking (OR 2.556, 95% CI 1.05-6.211, P=0.04). Irreversible airflow obstruction was present in 7%, and associated with ever smoking (OR 6.352, 95% CI 1.56-25.90, P=0.01). Impairment of diffusing capacity for carbon monoxide (DL_CO <70% predicted) was present in 57%, and associated with a history of previous tuberculosis (OR 2.01, 95% CI 1.00-4.00, P=0.47), but not with current or ever smoking. Conclusions: In this relatively young cohort of treated AIDS patients, a high proportion of whom were female, evidence of smoking-induced symptoms and airflow limitation was observed. The most common lung function abnormality, however, was an impairment in diffusing capacity, which was associated with previous tuberculosis. Further research to define structure/function relationships and the role of opportunistic infections and other risk factors is underway

Introduction The NYU Lung Cancer Biomarker Center recruited 1054 smokers (<=20 pack-years) between 03/2000 and 06/2010 and screened them with CT-scans and respiratory questionnaires. Questionnaires contained demographic characteristics, occupational exposures, lifestyle information including smoking history and alcohol use, personal and family medical history, and pulmonary symptoms. CT-scans showed the disease status and emphysema. We hypothesized that information collected in the questionnaires would be associated with the subject's disease status. Methods The 1054 Subjects were classified into 4 groups based on the results of enrollment CT-scans. This categorization showed 331 subjects with solid nodule(s) only, 95 with ground glass opacity (GGO) only, 126 with both solid nodule(s) and GGO(s), and the remaining 502 subjects with no nodules. Chi-squared tests were used to assess the relationship between disease category and each single predictor. Polytomous logistic regression was used to assess the association between disease category and multiple predictors. Forward/backward stepwise regression was used in model selection. Results Univariate analysis showed that gender (CMH =8.7092, p-value=0.0032), age (CMH=29.2219, p-value<0.0001), marijuana smoking (CMH=10.9024, p-value=0.0010), emphysema (CMH=9.0929, p-value=0.0026) and years of smoking (CMH=15.4712, p-value<0.0001) were significantly associated with disease categories. Further tests showed that years of smoking was highly associated with age, and did not contribute to prediction of disease category when age was included in the model, as shown by our multivariate analysis. In multivariate analysis, increasing age, gender (male) and history of emphysema were significant predictors of worsening disease class. Moreover, when we stratified by gender, the analysis indicated that in males, older men were more likely to develop solid nodules (OR=1.275, 95% CI [1.0063, 1.0490]) and more likely to have mixed nodules (OR= 1.0664, 95% CI= [1.0331, 1.1008]) than younger men. Males with emphysema were far more likely to have solid nodules compared with those without emphysema (OR= 1.9678, 95% CI= [1.3128, 2.9495]). Our analysis also revealed that in females, age was the only significant risk factor: older women were more likely to have mixed nodules than younger women (OR= 1.0593, 95% CI= [1.0274, 1.0922]). It is notable that all smoking related variables were not significantly associated with nodule class in our multivariable analysis. Conclusion Our analysis suggests that age, gender and emphysema play a critical role in the development of disease (solid nodule or GGO). Also, our analysis has shown that smoking is not likely to play a major role in the development of the disease in this high risk cohort

Alveolar macrophages (AMs) are exposed to respirable microbial particles. Similar to phagocytes in the gastrointestinal tract, AMs can suppress inflammation after exposure to nonpathogenic organisms. IL-1R-associated kinase-M (IRAK-M) is one inhibitor of innate immunity, normally suppressing pulmonary inflammation. During pneumonia, polymorphonuclear neutrophils (PMNs) are recruited by chemotactic factors released by AMs to produce an intense inflammation. We report that intact IRAK-M is strongly expressed in resting human AMs but is cleaved in patients with pneumonia via PMN-mediated induction of caspase-6 (CASP-6) activity. PMN contact is necessary and PMN membranes are sufficient for CASP-6 induction in macrophages. PMNs fail to induce TNF-alpha fully in macrophages expressing CASP-6 cleavage-resistant IRAK-M. Without CASP-6 expression, PMN stimulation fails to cleave IRAK-M, degrade IkappaBalpha, or induce TNF-alpha. CASP-6(-/-) mice subjected to cecal ligation and puncture have impaired TNF-alpha production in the lung and decreased mortality. LPS did not induce or require CASP-6 activity demonstrating that TLR2/4 signaling is independent from the CASP-6 regulated pathway. These data define a central role for CASP-6 in PMN-driven macrophage activation and identify IRAK-M as an important target for CASP-6. PMNs de-repress AMs via CASP-6-mediated IRAK-M cleavage. This regulatory system will blunt lung inflammation unless PMNs infiltrate the alveolar spaces

OBJECTIVES/SPECIFIC AIMS: The first year post 9/11/2001, the FEV1 of FDNY rescue workers declined 439 mL, stabilizing to a 25 ml/yr decline in the subsequent 7 years. Airflow obstruction predominated in firefighters who sought a subspecialty pulmonary evaluation for treatment. We are investigating the relationship between biomarkers of metabolic syndrome (MS) and decline in lung function. METHODS/ STUDY POPULATION: Treatment cohort (N = 1720) was stratified by FEV1 into obstructed, FEV1 < 76% predicted (LLN), or normal airflow, FEV1>76%. A pilot analysis assayed 41 patients' serum drawn 5 months post 9/11 for 15 biomarkers of MS by Luminex, (obstructed N = 10, normal N = 31). All patients had normal pre-9/11 lung function. Serum cholesterol (CHOL) and triglycerides (TG) were available on 157 patients, 20/157 were obstructed. Data presented as means +/- SD; p values -0.05 by t-test considered significant. RESULTS/ANTICIPATED RESULTS: BMIs at time of serum sampling were no different between normal and obstructed individuals. At subspecialty PFT, obstructed patients had higher BMIs with an accelerated decline in lung function post 9/11, increased airway reactivity, and evidence of air trapping based on elevated RV when compared to normals. Obstructed subjects had significantly greater CHOL and CHOL/HDL ratios; higher levels of sE-Selectin, tPAI-1, and s-ICAM; and a trend towards elevated levels of TG and C-peptide. DISCUSSION/SIGNIFICANCE OF IMPACT: Blood drawn post-WTC exposure identified a subgroup of patients with markers of MS. This subgroup had subsequent increased weight gain and decline in lung function. The finding of MS biomarkers prior to lung function decline raises the possibility that the combination of irritant exposure and mediators of MS interact and promote lung injury

Oxidants such as superoxide anion, hydrogen peroxide, and myeloperoxidase from activated inflammatory cells in the lower respiratory tract contribute to inflammation and injury. Etiologic agents include inorganic particulates such as asbestos, silica, or coal mine dust or mixtures of inorganic dust and combustion materials found in World Trade Center dust and smoke. These etiologic agents are phagocytosed by alveolar macrophages or bronchial epithelial cells and release chemotactic factors that recruit inflammatory cells to the lung. Chemotactic factors attract and activate neutrophils, eosinophils, mast cells, and lymphocytes and further activate macrophages to release more oxidants. Inorganic dusts target alveolar macrophages, World Trade Center dust targets bronchial epithelial cells, and eosinophils characterize tropical pulmonary eosinophilia (TPE) caused by filarial organisms. The technique of bronchoalveolar lavage in humans has recovered alveolar macrophages (AMs) in dust diseases and eosinophils in TPE that release increased amounts of oxidants in vitro. Interestingly, TPE has massively increased eosinophils in the acute form and after treatment can still have ongoing eosinophilic inflammation. A course of prednisone for one week can reduce the oxidant burden and attendant inflammation and may be a strategy to prevent chronic TPE and interstitial lung disease