Faculty Bibliography

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.

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)

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)