Faculty Bibliography

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.

Introduction: 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 microbiome of emphysema patients remains largely uncharacterized. We hypothesize that lung microbiota differs in early CT-defined emphysema subjects when compared with normal volunteers and that microbial load is associated with host cytokine production. Methods: Emphysema subjects were identified from the NYU Lung Cancer Center CT-scan screening cohort. Supraglotic and broncho-alveolar lavage (BAL) samples were obtained with bronchoscopy. Bacterial quantificationofepithelial lining fluid (ELF) was determined by qPCR using universal primers for eubacteria 16S rDNA. Bacteria speciation will be performed with 454 sequencing. Alveolar macrophages obtained after centrifugation of BAL were incubated with media alone or lipopolysaccharide (LPS). Thirty-ninecytokines were measured in BAL 24-hr supernatants using Luminex. Cytokine production was clustered based on the protein production post LPS stimulation compared with the media alone. Results: 24 subjects were enrolled (8 healthy normal volunteers and 16 with emphysema). Mean age was 37+/-10 and 62+/-6 yr. (p=0.001). All emphysema subjects were ex-smokers (normal 1+/-0.5 vs. 39+/-20 pack-yr., p=0.019). There were no differences in the %BAL cells between the two groups (Alveolar macrophages 89.5+/-4 vs. 88.1+/-8.2%). Lung function of emphysema subjects was characterized by lower FEV1/FVC (78.9+/-5.1 vs. 69.6+/-6.1, p=0.001) and hyperinflation (TLC=87.7+/-10.3 vs. 104.3+/-15.2% predicted, p=0.016). Bacteria rDNA was higher in the supraglotic area than lower respiratory tract (1.5e9+/-3.2e9 vs. 1e7+/-8.2e6 copies/mL of ELF, p<0.001). Bacterial load was similar in normal volunteers and emphysema subjects (8.4e6+/-8.8e6 vs. 1.1e7+/-8.1e6 copies/mL of ELF, p=ns). Cytokines'response to TLR stimulation grouped all study subjects into two clusters, and those with higher pro-inflammatory cytokine production were associated with higher amounts of bacteria in ELF (1.4e7+/-8.6e6 vs. 5.7e6+/-1.8e6 copies/mL, p<0.03). Conclusions: In the absence of signs or symptoms of infection both emphysema patients and normal controls had significant airway resident bacteria. Surprisingly, bacterial load was not increased in early CT-defined emphysema. Higher bacterial load was associated with higher TLR2/4-induced cytokine production. Next generation rDNA sequence data will define if microbiota is altered by emphysema or associated with TLR response

RATIONALE: The World Trade Center (WTC) destruction exposed ~13,000 FDNY rescue workers to aerosolized particulate matter. A subset of these patients developed obstructive lung disease. Matrix metalloproteinases(MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are important in tissue remodeling and are implicated in the development of airflow obstruction. Our work is focused on quantifying serum levels of these protease/antiproteases and determining their potential role in WTC-Lung Injury (WTC-LI). METHODS: In a nested case-control study of symptomatic non-smoking male firefighters with pre and post-9/11 spirometry data(N=1720), we defined cases (N=68) as having an FEV₁ at subspecialty pulmonary evaluation(SPE) in the lowest 12.5% of the cohort. Controls (N=119) were chosen from a random population stratified on BMI and FEV1at SPE. Serum drawn within six months of 9/11/2001, prior to the development of advanced WTC-LI, was assayed for MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, TIMP-1, TIMP-2, TIMP-3, and TIMP-4 by commercially available multiplex assays for Luminex(R&D Systems; Affymetrix). FEV₁ at SPE was correlated with serum MMP and TIMP. RESULTS: Cases and controls were compared by Pearson Chi-Square and Mann-Whitney U test where appropriate. Cases and controls were not significantly different in age, years of service, and WTC exposure intensity. Cases had significantly lower MMP-1 and MMP-3 than controls; Cases had elevated MMP-7, MMP-12, and MMP-13. MMP-2, MMP-8, MMP-9, TIMP-1, TIMP-2, TIMP-3, and TIMP-4 were not significantly different between cases and controls. At the time of SPE, cases had FEV_1%pred median(IQR) of 72(66-74), whereas controls had 96(88-104). Cases had a significantly lower DLCO, higher methacholine slope, and a greater number of subjects with a bronchodilator response compared to controls. There was no difference between cases and controls in airtrapping or in bronchial wall thickening by CT. Logistic regression modeling was used to determine the potential biomarkers' ability to predict WTC-LI. Elevated levels of MMP-12 had an odds ratio(OR) of being a case of 3.89, whereas elevated levels of MMP-3 and TIMP-3 had OR of being a control of 3.75 and 2.37. The model had Sensitivity of 48.5%, Specificity of 85.7% and a ROC Curve AUC of 0.757(95%CI:0.687-0.827), Figure 1. CONCLUSIONS: Serum levels of MMP and TIMP soon after 9/11 predicted lung function decline seven years later. The study finds that elevated MMP-12 is a risk factor for lung injury, whereas elevated MMP-3 and TIMP-3 are protective factors. These biomarkers of future lung injury may identify those most at risk for obstructive lung disease. (Figure Presented)

Introduction Measles is a highly contagious viral illness with significant mortality. Despite vaccination efforts, measles continues to occur in the United States. Most cases are associated with importation from endemic countries. Respiratory complications of the virus are one of the leading causes of fatalities. We report a case of measles pneumonia in an immunocompetent, unvaccinated adult with no associated importation from abroad. Case Presentation A 38 year old Caucasian female, with no recent travel history, presented with 3 days of fevers, cough, facial rash, and progressive shortness of breath. Her rash spread inferiorly to her trunk and extremities. On examination she was febrile, tachycardic, and hypoxic with a Pa02 of 55mmHg on room air. An erythematous, maculopapular rash was distributed over her face, trunk, and extremities. Her buccal mucosa had 2 mm white lesions, consistent with koplik spots. A chest x-ray showed a right upper lobe infiltrate and increased interstitial markings. She was placed in isolation and a floroquinolone was empirically initiated for a possible bacterial coinfection. Acute measles serologies returned positive, with an IgM level of 11.04 AU. Over the course of 48 hours, her fever defervesced, her oxygen saturation normalized, and her cough and rash resolved. She was discharged with instructions to have her 2 children receive the MMR vaccination immediately. Her children and she had not been vaccinated previously. Discussion Measles was declared eliminated from the United States in the year 2000, but the virus continues to be imported from endemic regions [1]. Our case represents one of 118 reports of measles in the United States between January 1 and May 20, 2011. Of these 118 cases, 105 (89%) were associated with importations from other countries and only 9 developed pneumonia [2]. Uniquely, our patient had no recent travel to, or contact with, individuals from an endemic region. This case, therefore, represents one of the few instances of measles pneumonia in the United States that was not import-associated. Instead, the risk factor in our presentation was the lack of prior vaccination. This absence of immunity resulted in the potentially life threatening complication of pneumonia. Our case underscores the importance of educating reluctant individuals regarding the safety and efficacy of the measles vaccine in preventing a devastating disease

RATIONALE: The destruction of the World Trade Center (WTC) led to the exposure of over 300,000 rescue workers and residents to particulate matter (WTC-PM) and other products of combustion. Victims have suffered from a wide array of adverse health effects. Particulates up to 50 microns have been recovered from the airways of rescue workers 10 months after the event. Our study focuses on understanding the cytokine and chemokines elaboration from the ex-vivo exposure of human alveolar macrophages (AM) from 15 subjects exposed to WTC-PM₅₃ and PM_2.5. METHODS: AM from 15 volunteers with normal chest radiographs were incubated in media alone (MA) to measure spontaneous cytokine release, LPS 40 ng/ml as a positive control, or suspensions of WTC-PM₅₃ and WTC-PM_2.5 at concentrations of 10, 50 and 100 mug/mL to assess the impact of WTC dust. Supernatants were assayed for 39 pro-inflammatory chemokines and cytokines 24 hours after exposure. RESULTS: PM₅₃ and PM_2.5 produced dose dependent analyte induction for a majority of the chemokines and cytokines. WTC-PM53 induced significantly more GM-CSF, G-CSF, IL-6, TNF-alpha, IL-10, IL-1beta, IP-10 and GRO than fine WTC-PM_2.5, Table 1. PM₅₃ 100mug/mL induced more GM-CSF than LPS 40ng/ml. For all other cytokines and chemokines tested PM₅₃ induced less analyte than LPS. Baseline GM-CSF and MDC elaboration varied in AM preparations; the median GM-CSF concentration was 16.4(IQR: 7-66) pg/mL, and MDC was 1803(483-3348). There was a strong correlation between baseline elaboration and dust induced GM-CSF and MDC; the correlation coefficients between baseline and dust-induced GM-CSF and MDC expression are shown in Table 2. CONCLUSIONS: Ex-vivo exposure of human AM to coarse WTC-PM53 stimulated production of significantly more inflammatory mediators than fine WTC-PM_2.5, suggesting that inhalation of coarse PM at the WTC produced high incidence of lung injury in those exposed. There was patient specific variation in response to dust that may contribute to individual susceptibility to lung injury after irritant dust exposure. (Table presented)

RATIONALE: The World Trade Center(WTC) collapse exposed over 300,000 rescue workers and residents to high amounts of particulate matter from debris. WTC-exposed FDNY rescue workers with pulmonary symptoms had primarily obstructive physiology. WTC exposure has also been linked to cardiovascular disease(CVD). Both CVD and COPD are characterized by systemic inflammation. Subjects with COPD are at greater risk for developing CVD. However the mechanism that links these two disease processes is poorly understood. Our goal was to quantify serum biomarkers of CVD present within six months of WTC-PM exposure. We then developed a predictive model that determines the odds of having WTC-Lung Injury(WTC-LI) within seven years after exposure. METHODS: A nested Case-Control design was used to identify the association of CVD biomarkers and development of WTC-LI post-9/11. All patients were non-smoking males, had normal pre-9/11 lung function, and had heart rate recorded at serum draw. Cases (N=61) were defined as FEV1%Pred in the lowest 12.5 percentile at the time of subspecialty pulmonary evaluation(SPE). Controls (N=109) were defined from a random sample that was enriched for those in the top octile of FEV1%Pred at SPE. We assayed serum collected within six months of 9/11 for biomarkers of CVD: Apolipoproteins(AI, AII, B, CII, CIII, E); C-Reactive Protein; Serum Amyloid A(SAA); Serum Amyloid P(SAP); Macrophage Inflammatory Protein-4(MIP-4, CCL18, PARC). RESULTS: Cases had significantly elevated Apolipoproteins(AI, AII, CII, CIII), SAA, and heart rate compared to controls. Controls had significantly elevated Apo E and MIP-4 compared to cases. Cutpoints were defined to optimize model parameters. The final logistic regression model determined the odds ratio(OR) of being a case using elevated heart rate (OR=3.5), elevated Apo AII(OR=6.8), CRP>3mg/L(OR=5.8), and lower levels of SAA(OR=17.9). Elevated levels of MIP-4 were seen as protective, and had an OR of being a control of 66.7. The model was adjusted for BMI at SPE, age at 9/11, exposure intensity and Pre-9/11 FEV1%Pred. The model had sensitivity of 74% and specificity of 85%. A ROC analysis determined an AUC of 0.891(95%CI:0.840-0.942), Figure 1. CONCLUSIONS: CVD biomarkers present in serum six months post-9/11 predict accelerated decline in lung function after exposure to WTC dust. The strong predictive ability of the model suggests that CVD biomarkers can also be used to predict lung injury. This suggests potential overlapping pathways of inflammation in CVD and lung injury in response to irritant exposure. (Figure Presented) Macrophage Inflammatory Protein-4; 3mg/L for CRP. The final logistic regression model used the cut points as predictors of airflow obstruction at subspecialty pulmonary evaluation. The models were adjusted for age, BMI, exposure intensity, and pre-9/11 FEV1% Predicted. The AUC of the final model was 0.891 (CI: 0.840-0.942). Sensitivity of the model is 74%; Specificity is 85%

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)

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!