Faculty Bibliography

The DNA replication protein Ciz1 promotes initiation of mammalian DNA replication in cooperation with cyclin A-dependent kinase, most likely by delivering cyclin A to sites where cyclin E-dependent pre-replication complex assembly has taken place. Normally, Ciz1 is anchored within nuclear matrix-associated foci that co-localize with sites of DNA replication, but in the absence of anchor domain Ciz1 retains the ability to promote initiation of DNA replication in isolated nuclei so attachment to the nuclear matrix is not essential for function. Expression of DNA replication and nuclear matrix anchor domains of Ciz1 are uncoupled and uneven at the transcript level in a wide range of common solid tumours, including breast and lung. In cell-based assays, recombinant anchor domain protein interferes with attachment of endogenous Ciz1 to the nuclear matrix, revealing a dominant negative effect that also impacts on nuclear matrix-recruitment of key components of the pre-replication complex. This suggests that Ciz1 normally plays a role in localizing initiation of DNA replication to the nuclear matrix. These findings implicate spatially unconstrained DNA replication as a source of nuclear disorder in cancer cells. We identified a variant Ciz1 isoform with alterations in the nuclear matrix attachment domain and tumour-restricted expression. RNAi-mediated selective inhibition of variant Ciz1 expression is sufficient to restrain the growth of tumour cells that express it, identifying variant Ciz1 as a functionally relevant driver of cell proliferation. We also present evidence that this form of Ciz1 is expressed in 34/35 lung tumours but not adjacent tissue, giving rise to stable protein quantifiable in less than a microlitre of patient plasma by western blot. Using two independent sets, with 170 and 160 samples, variant Ciz1 correctly identified stage 1 lung cancer patients with clinically useful accuracy. For set 1, mean variant Ciz1 level (+SD) in individuals without diagnosed tumours established a thre!

OBJECTIVES/SPECIFIC AIMS: The lung is classically thought to be sterile although molecular techniques for microbial identification are now suggesting the existence of a human airway microbiota. The study of the microbiome has now opened an opportunity to characterize resident microbial flora without the need for bacterial culture although the lung microbiome in smokers has not been characterized. We therefore tested the hypothesis that in smokers, the upper and lower airways contain a distinct microbiota. METHODS/STUDY POPULATION: We obtained supraglotic and broncho-alveolar lavage (BAL) samples in 8 subjects. Bacterial quantification of supraglotic aspirate and BAL was determined by qPCR using universal primers for 16S rDNA. Dilution was corrected by urea. We defined bacteria OTU by 454 sequencing. We performed cluster analysis, principal coordinate analysis and weighted UniFrac to determine microbiome. RESULTS/ANTICIPATED RESULTS: Subjects were 55 +/- 13 yo, 4/8 female and 7/8 significant smokers (>10 pack/year). FEV1 was 94 +/- 11%, FVC 108 +/- 10% and FEV1/FVC was 71 +/- 8. Bacteria rDNA was higher in the supraglotic area than BAL (1.5e9 +/- 3.2e9 vs. 1e7 +/- 8.2e6 copies/mL of adjusted fluid, p < 0.001). Clustering of the bacterial community at the family level showed distinct microbiome in the upper airway and the lung (BAL). While Prevotellaceae, Veillonellaceae and Fusobacteriaceae predominated in the supraglotic sample, Micrococcaceae, Propionibacteriaceae and Staphylococcaceae among others predominated in BAL. UniFrac calculated distances showed no overlapping circle of inertia between supraglotic and BAL (p < 0.0001 for first principal axis). DISCUSSION/SIGNIFICANCE OF IMPACT: In the absence of signs or symptoms of infection, subjects had significant airway resident bacteria. Th is supports the existence of a bacterial reservoir in the lung, which might be influenced by smoking and/or innate immunity

This document presents the proceedings from the American Thoracic Society Climate Change and Respiratory Health Workshop that was held on May 15, 2010, in New Orleans, Louisiana. The purpose of the one-day meeting was to address the threat to global respiratory health posed by climate change. Domestic and international experts as well as representatives of international respiratory societies and key U.S. federal agencies convened to identify necessary research questions concerning climate change and respiratory health and appropriate mechanisms and infrastructure needs for answering these questions. After much discussion, a breakout group compiled 27 recommendations for physicians, researchers, and policy makers. These recommendations are listed under main issues that the workshop participants deemed of key importance to respiratory health. Issues include the following: (1) the health impacts of climate change, with specific focus on the effect of heat waves, air pollution, and natural cycles; (2) mitigation and adaptation measures to be taken, with special emphasis on recommendations for the clinical and research community; (3) recognition of challenges specific to low-resource countries when coping with respiratory health and climate change; and (4) priority research infrastructure needs, with special discussion of international needs for cooperating with present and future environmental monitoring and alert systems.

RATIONALE: Cross-sectional studies demonstrate an association between metabolic syndrome and impaired lung function. OBJECTIVE: Define if metabolic syndrome biomarkers are risk factors for loss of lung function after irritant exposure. METHODS: A nested case-control study of FDNY personnel with normal pre-9/11 FEV1 and who presented for subspecialty pulmonary evaluation before 3/10/2008. We correlated metabolic syndrome biomarkers obtained within six months of World Trade Center Dust exposure with subsequent FEV1. FEV1 at subspecialty pulmonary evaluation within 6.5 years defined disease status; cases had FEV1<lower limit of normal (LLN) while controls had FEV1>/=LLN. MEASUREMENTS: Clinical data and serum sampled at the first monitoring exam within six months of 9/11/2001 assessed BMI, heart rate, serum glucose, Triglycerides/High Density Lipoprotein (HDL), Leptin, Pancreatic Polypeptide and Amylin. MAIN RESULTS: Cases and controls had significant differences in HDL<40 mg/dL with Triglycerides >/=150 mg/dL, heart rate >/=66 bpm, and Leptin >/=10,300 pg/mL. Each increased the odds of abnormal FEV1 at pulmonary evaluation by more than 2 fold, while Amylin >/=116 pg/mL decreased the odds by 84%, in a multi-biomarker model adjusting for age, race, BMI and WTC arrival time. This model had a sensitivity of 41%, a specificity of 86% and a ROC AUC of 0.77. CONCLUSION: Abnormal triglycerides and HDL, elevated heart rate and Leptin are independent risk factors of greater susceptibility to lung function impairment after 9/11/2001 while elevated Amylin is protective. Metabolic biomarkers are predictors of lung disease, and may be useful for assessing risk of impaired lung function in response to particulate inhalation

RATIONALE: Biomarkers of sepsis severity and mortality are an important area of investigation. Our group has shown that increased serum levels of soluble(s)CD28 and sCD40L are predictive of mortality from sepsis. This has more recently been validated by other groups. We hypothesize that the release of microparticles(MPs) bound to co-stimulatory molecules into the circulation is one of the causes of disseminated inflammation. The mechanism producing sCD28 and sCD40L is unknown. We have previously shown that MP activity is induced in murine sepsis. Our current work is focused on using flow-cytometry to identify the characteristics of MPs and determine if they express CD28 and CD40L in sepsis models. METHODS: in vivo: Polymicrobial sepsis was induced in C57BL/6 mice using 19-Gauge cecal ligation and puncture(CLP) and citrated plasma was collected by cardiac puncture 18 hours later. in vitro: RAW264.7 cells 2x106 cells/mL in a 6 well plate were exposed to LPS(40 ng/mL) or media alone and incubated overnight. MP Isolation: MP containing supernatants were centrifuged at 1,500gX15 min and ultracentrifugation at 70,000gX30 min. Flow Cytometric Analysis was performed to quantify MP populations in the in vivo and in vitro models (AccuriC6). Forward scatter(FSC) and side scatter(SSC) of light was set in logarithmic scale, and the threshold was set at the FSC parameter(FS=1). The fluorescence channels were also set at logarithmic gain. Standard fluorescent microbeads(1 mum) were used to set the MP gate. In order to distinguish true events from electronic noise and increase the specificity of microparticle detection, events in the MP gate were further discriminated by labeling with PE-Annexin-V. MP expression of CD28/CD40L was determined using APC-labeled CD28 and PerCP-labeled CD40L. RESULTS: MPs were imaged using TEM, Figure 1. MPs were identified and characterized using flow cytometry, Figure 2A-B. Plasma from operated septic mice had 2.5-fold greater Annexin-V+ MPs compared to unoperated non-septic controls, Figure 2C. Annexin-V+ MPs were 3.5-fold greater in RAW cells exposed to LPS as compared to control. We have observed MPs expressing CD28 and CD40L in the serum of septic mice. CONCLUSIONS: MP counts are higher in both in vivo and in vitro models of sepsis as compared to unoperated non-septic controls. Preliminary data shows that MPs may be carriers of biologically active CD28 and CD40L. Future experiments will further delineate inflammatory mediators expressed on the surface of MPs and their role in sepsis. (Figure Presented)

BACKGROUND: Low-dose computed tomography (CT) for lung cancer screening can reduce lung cancer mortality. The National Lung Screening Trial reported a 20% reduction in lung cancer mortality in high-risk smokers. However, CT scanning is extremely sensitive and detects non-calcified nodules (NCNs) in 24-50% of subjects, suggesting an unacceptably high false-positive rate. We hypothesized that by reviewing demographic, clinical and nodule characteristics, we could identify risk factors associated with the presence of nodules on screening CT, and with the probability that a NCN was malignant. METHODS: We performed a longitudinal lung cancer biomarker discovery trial (NYU LCBC) that included low-dose CT-screening of high-risk individuals over 50 years of age, with more than 20 pack-year smoking histories, living in an urban setting, and with a potential for asbestos exposure. We used case-control studies to identify risk factors associated with the presence of nodules (n = 625) versus no nodules (n = 557), and lung cancer patients (n = 30) versus benign nodules (n = 128). RESULTS: The NYU LCBC followed 1182 study subjects prospectively over a 10-year period. We found 52% to have NCNs >4 mm on their baseline screen. Most of the nodules were stable, and 9.7% of solid and 26.2% of sub-solid nodules resolved. We diagnosed 30 lung cancers, 26 stage I. Three patients had synchronous primary lung cancers or multifocal disease. Thus, there were 33 lung cancers: 10 incident, and 23 prevalent. A sub-group of the prevalent group were stable for a prolonged period prior to diagnosis. These were all stage I at diagnosis and 12/13 were adenocarcinomas. CONCLUSIONS: NCNs are common among CT-screened high-risk subjects and can often be managed conservatively. Risk factors for malignancy included increasing age, size and number of nodules, reduced FEV1 and FVC, and increased pack-years smoking. A sub-group of screen-detected cancers are slow-growing and may contribute to over-diagnosis and lead-time biases.

BACKGROUND: The WTC collapse exposed over 300,000 people to high concentrations of WTC-PM; particulates up to approximately 50 mm were recovered from rescue workers' lungs. Elevated MDC and GM-CSF independently predicted subsequent lung injury in WTC-PM-exposed workers. Our hypotheses are that components of WTC dust strongly induce GM-CSF and MDC in AM; and that these two risk factors are in separate inflammatory pathways. METHODOLOGY/PRINCIPAL FINDINGS: Normal adherent AM from 15 subjects without WTC-exposure were incubated in media alone, LPS 40 ng/mL, or suspensions of WTC-PM(10-53) or WTC-PM(2.5) at concentrations of 10, 50 or 100 microg/mL for 24 hours; supernatants assayed for 39 chemokines/cytokines. In addition, sera from WTC-exposed subjects who developed lung injury were assayed for the same cytokines. In the in vitro studies, cytokines formed two clusters with GM-CSF and MDC as a result of PM(10-53) and PM(2.5). GM-CSF clustered with IL-6 and IL-12(p70) at baseline, after exposure to WTC-PM(10-53) and in sera of WTC dust-exposed subjects (n = 70) with WTC lung injury. Similarly, MDC clustered with GRO and MCP-1. WTC-PM(10-53) consistently induced more cytokine release than WTC-PM(2.5) at 100 microg/mL. Individual baseline expression correlated with WTC-PM-induced GM-CSF and MDC. CONCLUSIONS: WTC-PM(10-53) induced a stronger inflammatory response by human AM than WTC-PM(2.5). This large particle exposure may have contributed to the high incidence of lung injury in those exposed to particles at the WTC site. GM-CSF and MDC consistently cluster separately, suggesting a role for differential cytokine release in WTC-PM injury. Subject-specific response to WTC-PM may underlie individual susceptibility to lung injury after irritant dust exposure.

INTRODUCTION: As CT screening is integrated into non-small cell lung cancer (NSCLC) care, additional parameters are needed to help distinguish cancers from benign nodules. Osteopontin (OPN), a secreted phosphoprotein, has elevated plasma levels in NSCLC. We hypothesize that changes in plasma OPN over time (i.e., OPN velocity [OPNV]) can differentiate NSCLC patients from those without cancer in a CT screening population. METHODS: A nested case-control study was conducted within a NSCLC CT screening trial. Incident cancers with serial plasma were matched to controls. OPN was measured by ELISA. Demographic, OPN, and OPNV were compared between cancers and controls using Wilcoxon Signed Rank tests. RESULTS: Ten incident cancers were identified. The pack years distributions were similar, but cancers were older (median of the paired difference: 5.35 years; p=0.002) and their surveillance intervals were shorter (median of the paired difference: -2 months; p=0. 03) than matched controls. Baseline OPN was similar (median of the paired difference: -5.15 ng/ml, p=0.50), but OPNV in the cancers was significantly greater than that of matched controls, (median of the paired difference: 1.06 ng/ml/month, p=0.01). Accuracy rate for prediction of disease status based on OPNV (adjusted for age and surveillance) was 83%. CONCLUSIONS: These are early evidence for utility of monitoring plasma OPN during CT screening to assist in identification of NSCLCs.