Philip A Bromberg

Publications of Philip A Bromberg

• Linking oxidative events to inflammatory and adaptive gene expression induced by exposure to an organic particulate matter component.

  Authors: Wan-Yun Cheng, Jenna Currier, Philip A Bromberg, Robert Silbajoris, Steven O Simmons, James M Samet

  Environmental health perspectives. 02/2012; 120(2):267-74.

  Toxicological studies have correlated inflammatory effects of diesel exhaust particles (DEP) with its organic constituents, such as the organic electrophile 1,2-naphthoquinone (1,2-NQ). To elucidateToxicological studies have correlated inflammatory effects of diesel exhaust particles (DEP) with its organic constituents, such as the organic electrophile 1,2-naphthoquinone (1,2-NQ). To elucidate the mechanisms involved in 1,2-NQ-induced inflammatory responses, we examined the role of oxidant stress in 1,2-NQ-induced expression of inflammatory and adaptive genes in a human airway epithelial cell line. We measured cytosolic redox status and hydrogen peroxide (H2O2) in living cells using the genetically encoded green fluorescent protein (GFP)-based fluorescent indicators roGFP2 and HyPer, respectively. Expression of interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), and heme oxygenase-1 (HO-1) mRNA was measured in BEAS-2B cells exposed to 1,2-NQ for 1-4 hr. Catalase overexpression and metabolic inhibitors were used to determine the role of redox changes and H2O2 in 1,2-NQ-induced gene expression. Cells expressing roGFP2 and HyPer showed a rapid loss of redox potential and an increase in H2O2 of mitochondrial origin following exposure to 1,2-NQ. Overexpression of catalase diminished the H2O2-dependent signal but not the 1,2-NQ-induced loss of reducing potential. Catalase overexpression and inhibitors of mitochondrial respiration diminished elevations in IL-8 and COX-2 induced by exposure to 1,2-NQ, but potentiated HO-1 mRNA levels in BEAS cells. These data show that 1,2-NQ exposure induces mitochondrial production of H2O2 that mediates the expression of inflammatory genes, but not the concurrent loss of reducing redox potential in BEAS cells. 1,2-NQ exposure also causes marked expression of HO-1 that appears to be enhanced by suppression of H2O2. These findings shed light into the oxidant-dependent events that underlie cellular responses to environmental electrophiles.

• Ambient particulate matter induces interleukin-8 expression through an alternative NF-κB (nuclear factor-kappa B) mechanism in human airway epithelial cells.

  Authors: Robert Silbajoris, Alvaro R Osornio-Vargas, Steven O Simmons, William Reed, Philip A Bromberg, Lisa A Dailey, James M Samet

  Environmental health perspectives. 06/2011; 119(10):1379-83.

  Exposure to ambient air particulate matter (PM) has been shown to increase rates of cardiopulmonary morbidity and mortality, but the underlying mechanisms are still not well understood.Objective: WeExposure to ambient air particulate matter (PM) has been shown to increase rates of cardiopulmonary morbidity and mortality, but the underlying mechanisms are still not well understood.Objective: We examined signaling events involved in the expression of the inflammatory gene interleukin-8 (IL-8) in human airway epithelial cells (HAECs) exposed to ambient PM collected in an urban area of Mexicali, Mexico. We studied IL-8 expression and regulatory signaling pathways in cultured HAECs exposed to Mexicali PM suspended in media for 0-4 hr. Exposure resulted in a dose-dependent, 2- to 8-fold increase in IL-8 mRNA expression relative to controls. PM exposure induced IL-8 transcriptional activity in BEAS-2B cells that was dependent on the nuclear factor-kappa B (NF-κB) response element in the IL-8 promoter. Chromatin immunoprecipitation (ChIP) assays showed a 3-fold increase in binding of the p65 (RelA) NF-κB isoform to the IL-8 promoter sequence in HAECs exposed to PM. Western blot analyses showed elevated levels of phosphorylation of p65 but no changes in IκBα phosphorylation or degradation. IL-8 expression was blunted in a dose-dependent manner in BEAS-2B cells transduced with a lentivirus encoding a dominant negative p65 mutant in which phosphorylation sites were inactivated. Taken together, these findings show that the increase in IL-8 mRNA expression in HAECs exposed to PM10 (PM ≤ 10 μm in aerodynamic diameter) is mediated through an NF-κB-dependent signaling mechanism that occurs through a pathway involving direct phosphorylation of the transcription factor p65 in the absence of IκBα degradation. These data show that exposure to PM10 in ambient air can induce inflammatory responses by activating specific signaling mechanisms in HAECs.

• Enhancement of systemic and sputum granulocyte response to inhaled endotoxin in people with the GSTM1 null genotype.

  Authors: Madeline A Dillon, Bradford Harris, Michelle L Hernandez, Baiming Zou, William Reed, Philip A Bromberg, Robert B Devlin, David Diaz-Sanchez, Steven Kleeberger, Haibo Zhou, John C Lay, Neil E Alexis, David B Peden

  Occupational and environmental medicine. 03/2011; 68(10):783-5.

  To determine if the GSTM1 null genotype is a risk factor for increased inflammatory response to inhaled endotoxin. 35 volunteers who had undergone inhalation challenge with a 20 000 endotoxin unitTo determine if the GSTM1 null genotype is a risk factor for increased inflammatory response to inhaled endotoxin. 35 volunteers who had undergone inhalation challenge with a 20 000 endotoxin unit dose of Clinical Center Reference Endotoxin (CCRE) were genotyped for the GSTM1 null polymorphism. Parameters of airway and systemic inflammation observed before and after challenge were compared in GSTM1 null (n=17) and GSTM1 (n=18) sufficient volunteers. GSTM1 null volunteers had significantly increased circulating white blood cells (WBCs), polymorphonuclear neutrophils (PMNs), platelets and sputum PMNs (% sputum PMNs and PMNs/mg sputum) after CCRE challenge. GSTM1 sufficient volunteers had significant, but lower increases in circulating WBCs, PMNs and % sputum PMNs, and no increase in platelets or PMNs/mg sputum. Linear regression analysis adjusted for baseline values of the entire cohort revealed that the GSTM1 null genotype significantly increased circulating WBCs, platelets and % sputum PMNs after challenge. These data support the hypothesis that the GSTM1 null genotype is a risk factor for increased acute respiratory and systemic inflammatory response to inhaled CCRE. These data are consistent with other observations that the GSTM1 null genotype is associated with increased respiratory, systemic and cardiovascular effects linked to ambient air particulate matter exposure and indicate that the GSTM1 null genotype should be considered a risk factor for adverse health effects associated with exposure to environmental endotoxin.

• Darkfield-confocal microscopy detection of nanoscale particle internalization by human lung cells.

  Authors: Eugene A Gibbs-Flournoy, Philip A Bromberg, Thomas P J Hofer, James M Samet, Robert M Zucker

  Particle and fibre toxicology. 01/2011; 8(1):2.

  Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately,Concerns over the health effects of nanomaterials in the environment have created a need for microscopy methods capable of examining the biological interactions of nanoparticles (NP). Unfortunately, NP are beyond the diffraction limit of resolution for conventional light microscopy (~200 nm). Fluorescence and electron microscopy techniques commonly used to examine NP interactions with biological substrates have drawbacks that limit their usefulness in toxicological investigation of NP. EM is labor intensive and slow, while fluorescence carries the risk of photobleaching the sample and has size resolution limits. In addition, many relevant particles lack intrinsic fluorescence and therefore can not be detected in this manner. To surmount these limitations, we evaluated the potential of a novel combination of darkfield and confocal laser scanning microscopy (DF-CLSM) for the efficient 3D detection of NP in human lung cells. The DF-CLSM approach utilizes the contrast enhancements of darkfield microscopy to detect objects below the diffraction limit of 200 nm based on their light scattering properties and interfaces it with the power of confocal microscopy to resolve objects in the z-plane. Validation of the DF-CLSM method using fluorescent polystyrene beads demonstrated spatial colocalization of particle fluorescence (Confocal) and scattered transmitted light (Darkfield) along the X, Y, and Z axes. DF-CLSM imaging was able to detect and provide reasonable spatial locations of 27 nm TiO2 particles in relation to the stained nuclei of exposed BEAS 2B cells. Statistical analysis of particle proximity to cellular nuclei determined a significant difference between 5 min and 2 hr particle exposures suggesting a time-dependent internalization process. DF-CLSM microscopy is an alternative to current conventional light and electron microscopy methods that does not rely on particle fluorescence or contrast in electron density. DF-CLSM is especially well suited to the task of establishing the spatial localization of nanoparticles within cells, a critical topic in nanotoxicology. This technique has advantages to 2D darkfield microscopy as it visualizes nanoparticles in 3D using confocal microscopy. Use of this technique should aid toxicological studies related to observation of NP interactions with biological endpoints at cellular and subcellular levels.

• Atopic asthmatic subjects but not atopic subjects without asthma have enhanced inflammatory response to ozone.

  Authors: Michelle L Hernandez, John C Lay, Bradford Harris, Charles R Esther, W June Brickey, Philip A Bromberg, David Diaz-Sanchez, Robert B Devlin, Steven R Kleeberger, Neil E Alexis, David B Peden

  The Journal of allergy and clinical immunology. 09/2010; 126(3):537-44.e1.

  Asthma is a known risk factor for acute ozone-associated respiratory disease. Ozone causes an immediate decrease in lung function and increased airway inflammation. The role of atopy and asthma inAsthma is a known risk factor for acute ozone-associated respiratory disease. Ozone causes an immediate decrease in lung function and increased airway inflammation. The role of atopy and asthma in modulation of ozone-induced inflammation has not been determined. We sought to determine whether atopic status modulates ozone response phenotypes in human subjects. Fifty volunteers (25 healthy volunteers, 14 atopic nonasthmatic subjects, and 11 atopic asthmatic subjects not requiring maintenance therapy) underwent a 0.4-ppm ozone exposure protocol. Ozone response was determined based on changes in lung function and induced sputum composition, including airway inflammatory cell concentration, cell-surface markers, and cytokine and hyaluronic acid concentrations. All cohorts experienced similar decreases in lung function after ozone. Atopic and atopic asthmatic subjects had increased sputum neutrophil numbers and IL-8 levels after ozone exposure; values did not significantly change in healthy volunteers. After ozone exposure, atopic asthmatic subjects had significantly increased sputum IL-6 and IL-1beta levels and airway macrophage Toll-like receptor 4, Fc(epsilon)RI, and CD23 expression; values in healthy volunteers and atopic nonasthmatic subjects showed no significant change. Atopic asthmatic subjects had significantly decreased IL-10 levels at baseline compared with healthy volunteers; IL-10 levels did not significantly change in any group with ozone. All groups had similar levels of hyaluronic acid at baseline, with increased levels after ozone exposure in atopic and atopic asthmatic subjects. Atopic asthmatic subjects have increased airway inflammatory responses to ozone. Increased Toll-like receptor 4 expression suggests a potential pathway through which ozone generates the inflammatory response in allergic asthmatic subjects but not in atopic subjects without asthma.

• Comparative airway inflammatory response of normal volunteers to ozone and lipopolysaccharide challenge.

  Authors: Michelle L Hernandez, Bradford Harris, John C Lay, Philip A Bromberg, David Diaz-Sanchez, Robert B Devlin, Steven R Kleeberger, Neil E Alexis, David B Peden

  Inhalation toxicology. 07/2010; 22(8):648-56.

  Ozone and lipopolysaccharide (LPS) are environmental pollutants with adverse health effects noted in both healthy and asthmatic individuals. The authors and others have shown that inhalation of ozoneOzone and lipopolysaccharide (LPS) are environmental pollutants with adverse health effects noted in both healthy and asthmatic individuals. The authors and others have shown that inhalation of ozone and LPS both induce airway neutrophilia. Based on these similarities, the authors tested the hypothesis that common biological factors determine response to these two different agents. Fifteen healthy, nonasthmatic volunteers underwent a 0.4 part per million ozone exposure for 2 h while performing intermittent moderate exercise. These same subjects underwent an inhaled LPS challenge with 20,000 LPS units of Clinical Center Reference LPS, with a minimum of 1 month separating these two challenge sessions. Induced sputum was obtained 24 h before and 4-6 h after each exposure session. Sputum was assessed for total and differential cell counts and expression of cell surface proteins as measured by flow cytometry. Sputum supernatants were assayed for cytokine concentration. Both ozone and LPS challenge augmented sputum neutrophils and subjects' responses were significantly correlated (R = .73) with each other. Ozone had greater overall influence on cell surface proteins by modifying both monocytes (CD14, human leukocyte antigen [HLA]-DR, CD11b) and macrophages (CD11b, HLA-DR) versus LPS where CD14 and HLA-DR were modified only on monocytes. However, LPS significantly increased interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, with no significant increases seen after ozone challenge. Ozone and LPS exposure in healthy volunteers induce similar neutrophil responses in the airways; however, downstream activation of innate immune responses differ, suggesting that oxidant versus bacterial air pollutants may be mediated by different mechanisms.

• Low-level ozone exposure induces airways inflammation and modifies cell surface phenotypes in healthy humans.

  Authors: Neil E Alexis, John C Lay, Milan Hazucha, Bradford Harris, Michelle L Hernandez, Philip A Bromberg, Howard Kehrl, David Diaz-Sanchez, Chong Kim, Robert B Devlin, David B Peden

  Inhalation toxicology. 04/2010; 22(7):593-600.

  The effects of low-level ozone exposure (0.08 ppm) on pulmonary function in healthy young adults are well known; however, much less is known about the inflammatory and immunomodulatory effects ofThe effects of low-level ozone exposure (0.08 ppm) on pulmonary function in healthy young adults are well known; however, much less is known about the inflammatory and immunomodulatory effects of low-level ozone in the airways. Techniques such as induced sputum and flow cytometry make it possible to examine airways inflammatory responses and changes in immune cell surface phenotypes following low-level ozone exposure. The purpose of this study was to determine if exposure to 0.08 parts per million ozone for 6.6 h induces inflammation and modifies immune cell surface phenotypes in the airways of healthy adult subjects. Fifteen normal volunteers underwent an established 0.08 part per million ozone exposure protocol to characterize the effect of ozone on airways inflammation and immune cell surface phenotypes. Induced sputum and flow cytometry were used to assess these endpoints 24 h before and 18 h after exposure. The results showed that exposure to 0.08 ppm ozone for 6.6 h induced increased airway neutrophils, monocytes, and dendritic cells and modified the expression of CD14, HLA-DR, CD80, and CD86 on monocytes 18 h following exposure. Exposure to 0.08 parts per million ozone is associated with increased airways inflammation and promotion of antigen-presenting cell phenotypes 18 hours following exposure. These findings need to be replicated in a similar experiment that includes a control air exposure.

• An integrated imaging approach to the study of oxidative stress generation by mitochondrial dysfunction in living cells.

  Authors: Wan-Yun Cheng, Haiyan Tong, Evan W Miller, Christopher J Chang, James Remington, Robert M Zucker, Philip A Bromberg, James M Samet, Thomas P J Hofer

  Environmental health perspectives. 04/2010; 118(7):902-8.

  The mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has beenThe mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has been implicated in a variety of oxidant-dependent toxicological responses. Unlike ions of other transition metals such as iron, copper, and vanadium, Zn(2+) does not generate reactive oxygen species (ROS) through redox cycling. To characterize the role of oxidative stress in zinc-induced toxicity. We used an integrated imaging approach that employs the hydrogen peroxide (H2O2)-specific fluorophore Peroxy Green 1 (PG1), the mitochondrial potential sensor 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and the mitochondria-targeted form of the redox-sensitive genetically encoded fluorophore MTroGFP1 in living cells. Zinc treatment in the presence of the Zn(2+) ionophore pyrithione of A431 skin carcinoma cells preloaded with the H(2)O(2)-specific indicator PG1 resulted in a significant increase in H(2)O(2) production that could be significantly inhibited with the mitochondrial inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as the source of zinc-induced H(2)O(2) formation by the observation that exposure to zinc caused a loss of mitochondrial membrane potential. Using MTroGFP1, we showed that zinc exposure of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from mouse hearts. Taken together, these findings show a disruption of mitochondrial integrity, H(2)O(2) formation, and a shift toward positive redox potential in cells exposed to zinc. These data demonstrate the utility of real-time, live-cell imaging to study the role of oxidative stress in toxicological responses.

• Phosphorylation of p65 is required for zinc oxide nanoparticle-induced interleukin 8 expression in human bronchial epithelial cells.

  Authors: Weidong Wu, James M Samet, David B Peden, Philip A Bromberg

  Environmental health perspectives. 03/2010; 118(7):982-7.

  Exposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as interleukin-8 (IL-8). WeExposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as interleukin-8 (IL-8). We investigated the effect of ZnO nanoparticles on IL-8 expression and the underlying mechanisms in human bronchial epithelial cells. We determined IL-8 mRNA and protein expression in primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line using reverse-transcriptase polymerase chain reaction and the enzyme-linked immunosorbent assay, respectively. Transcriptional activity of IL-8 promoter and nuclear factor kappa B (NFkappaB) in ZnO-treated BEAS-2B cells was measured using transient gene transfection of the luciferase reporter construct with or without p65 constructs. Phosphorylation and degradation of IkappaBalpha, an inhibitor of NF-kappaB, and phosphorylation of p65 were detected using immunoblotting. Binding of p65 to the IL-8 promoter was examined using the chromatin immunoprecipitation assay. ZnO exposure (2-8 microg/mL) increased IL-8 mRNA and protein expression. Inhibition of transcription with actinomycin D blocked ZnO-induced IL-8 expression, which was consistent with the observation that ZnO exposure increased IL-8 promoter reporter activity. Further study demonstrated that the kappaB-binding site in the IL-8 promoter was required for ZnO-induced IL-8 transcriptional activation. ZnO stimulation modestly elevated IkappaBalpha phosphorylation and degradation. Moreover, ZnO exposure also increased the binding of p65 to the IL-8 promoter and p65 phosphorylation at serines 276 and 536. Overexpression of p65 constructs mutated at serines 276 or 536 significantly reduced ZnO-induced increase in IL-8 promoter reporter activity. p65 phosphorylation and IkappaBalpha phosphorylation and degradation are the primary mechanisms involved in ZnO nanoparticle-induced IL-8 expression in human bronchial epithelial cells.

• The glutathione-S-transferase Mu 1 null genotype modulates ozone-induced airway inflammation in human subjects.

  Authors: Neil E Alexis, Haibo Zhou, John C Lay, Bradford Harris, Michelle L Hernandez, Tsui-Shan Lu, Philip A Bromberg, David Diaz-Sanchez, Robert B Devlin, Steven R Kleeberger, David B Peden

  The Journal of allergy and clinical immunology. 09/2009;

  BACKGROUND: The glutathione-S-transferase Mu 1 (GSTM1) null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone levels. OzoneBACKGROUND: The glutathione-S-transferase Mu 1 (GSTM1) null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone levels. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. However, it is not known whether GSTM1 modulates these ozone responses in vivo in human subjects. OBJECTIVE: The purpose of this study was to determine whether the GSTM1 null genotype modulates ozone responses in human subjects. METHODS: Thirty-five healthy volunteers were genotyped for the GSTM1 null mutation and underwent a standard ozone exposure protocol to determine whether lung function and inflammatory responses to ozone were different between the 19 GSTM1 wild type and 16 GSTM1 null volunteers. RESULTS: GSTM1 did not modulate lung function responses to acute ozone. Granulocyte influx 4 hours after challenge was similar between GSTM1 normal and null volunteers. However, GSTM1 null volunteers had significantly increased airway neutrophils 24 hours after challenge, as well as increased expression of HLA-DR on airway macrophages and dendritic cells. CONCLUSION: The GSTM1 null genotype is associated with increased airways inflammation 24 hours after ozone exposure, which is consistent with the lag time observed between increased ambient air ozone exposure and exacerbations of lung disease.

• Diesel Particle-induced Transcription Expression of P21 Involves Activation of EGFR, SRC and STAT3.

  Authors: Dongsun Cao, Philip A Bromberg, James M Samet

  American journal of respiratory cell and molecular biology. 04/2009;

  Exposure to diesel exhaust particle has been associated with adverse health outcomes, such as inflammation, adjuvancy and mutagenesis. However, the molecular mechanisms by which DEP inhalation exertsExposure to diesel exhaust particle has been associated with adverse health outcomes, such as inflammation, adjuvancy and mutagenesis. However, the molecular mechanisms by which DEP inhalation exerts these effects are still largely unknown. We previously reported that exposure to DEP activates the transcription factor Stat3 in airway epithelial cells, a primary target cell of inhaled DEP. To elucidate the functional role of Stat3 activation in these cells, we investigated the function of Stat3 in DEPâinduced expression of the p21 gene in the human bronchial epithelial cell line BEAS-2B. We report that DEP exposure induces increased levels of p21 mRNA and protein in a manner that is independent of p53 and Sp1 expression or DNA binding to the p21 gene. Using Chromatin Immuoprecipitation assays and expression of a dominant negative Stat3 mutant, we show that activation of Stat3 and its binding to the p21 promoter are required for DEP-induced expression of p21, suggesting that Stat3 plays an essential role in the induction of p21 by DEP. Additional experiments demonstrated that activation of p21 gene expression is dependent on the activation of EGFR and Src kinase activities. Lastly, we provide evidence suggesting that DEP exposure can inhibit the proliferation of human bronchial epithelial cells, suggesting a functional role of p21 activation airway epithelial cells exposed to DEP.

• Influence of C-159T SNP of the CD14 gene promoter on lung function in smokers.

  Authors: Haibo Zhou, Neil E Alexis, Martha Almond, James Donohue, Craig Laforce, Philip A Bromberg, David B Peden

  Respiratory medicine. 04/2009;

  CD14, a co-receptor for endotoxin, plays a significant role in regulating the inflammatory response to this agent. The C-159T single nucleotide polymorphism (SNP) in the CD14 gene promoter is anCD14, a co-receptor for endotoxin, plays a significant role in regulating the inflammatory response to this agent. The C-159T single nucleotide polymorphism (SNP) in the CD14 gene promoter is an important regulator of CD14 expression, with TT homozygotes having increased expression of CD14. This SNP has been linked to pathogenesis of asthma and with cardiovascular diseases in smokers. We hypothesize that CD14 also plays a role in the pathophysiology of COPD in smokers who are exposed to endotoxin contained in cigarette smoke as well as endotoxin derived from Gram-negative microbes colonizing their airways. To assess the effect of the C-159T SNP of the CD14 gene promoter on lung function, we recruited 246 smokers 40 years of age or older with a range of 10-156 pack-year smoking exposures. The TT genotype was associated with lower lung function in smokers with a moderate smoking history. However, the CC genotype was associated with decreased lung function in heavy smokers (>56 pack years). The effect of CC genotype on severity of COPD is analogous with the effect of this genotype in risk for asthma. CD14 may be a factor in the pathophysiology of COPD, as it is in asthma and smoking-related cardiovascular diseases.

• Concentrated Ambient Ultrafine Particle Exposure Induces Cardiac Changes in Young Healthy Volunteers.

  Authors: James M Samet, Ana Rappold, Donald Graff, Wayne E Cascio, Jon H Berntsen, Yuh-Chin T Huang, Margaret Herbst, Maryann Bassett, Tracey Montilla, Milan J Hazucha, Philip A Bromberg, Robert B Devlin

  American journal of respiratory and critical care medicine. 03/2009;

  RATIONALE: Exposure to ambient ultrafine particles has been associated with cardiopulmonary toxicity and mortality. Adverse effects specifically linked to ultrafine particles include loss ofRATIONALE: Exposure to ambient ultrafine particles has been associated with cardiopulmonary toxicity and mortality. Adverse effects specifically linked to ultrafine particles include loss of sympathovagal balance and altered hemostasis. OBJECTIVE: To characterize the effects of acute exposure to ambient ultrafine particle in young healthy humans METHODS: Twenty healthy non-smoking male and female subjects between the ages of 18 and 35 were exposed to filtered air or to an atmosphere in which captured ultrafine (<0.16 um) particles were concentrated by a factor of up to 20 fold over ambient levels with the use of particle concentrators fitted with size-selective outlets (UFCAP). Subjects underwent bronchoalveolar lavage 18 hrs following each exposure. Cardiovascular endpoints measured included pulmonary function, clinical chemistry and hematological parameters as well as heart rate variability and repolarization indices. MEASUREMENTS AND MAIN RESULTS: Exposure to UFCAP was statistically associated with an increase in frequency domain markers of heart rate variability, specifically indicative of elevated vagal input to the heart. Consistent with this finding were increases in the variance associated with the duration of the QT interval. In addition, UFCAP exposure resulted in a significant increase in blood levels of the fibrin degradation product D-dimer as well as a modest elevation in the inflammatory chemokine IL-8 recovered in the lavage fluid. CONCLUSIONS: These findings show mild inflammatory and pro-thrombic responses, and are suggestive of alterations in cardiac repolarization induced by UFCAP inhalation.

• Mechanisms of LPS-induced CD40 expression in human peripheral blood monocytic cells.

  Authors: Weidong Wu, Neil E Alexis, Philip A Bromberg, Ilona Jaspers, David B Peden

  Biochemical and biophysical research communications. 01/2009;

  CD40 plays important roles in cell-mediated and humoral immune responses. In this study, we explored mechanisms underlying lipopolysaccharide (LPS)-induced CD40 expression in purified humanCD40 plays important roles in cell-mediated and humoral immune responses. In this study, we explored mechanisms underlying lipopolysaccharide (LPS)-induced CD40 expression in purified human peripheral blood monocytic cells (PBMCs) from healthy volunteers. Exposure to LPS induced increases in CD40 mRNA and protein expression on PBMCs. LPS stimulation caused IkappaBalpha degradation. Inhibition of NFkappaB activation abrogated LPS-induced CD40 expression. LPS stimulation also resulted in phosphorylation of mitogen-activated protein kinases, however, only Jun N-terminal kinase (JNK) was partially involved in LPS-induced CD40 expression. In addition, LPS exposure resulted in elevated interferon gamma (IFNgamma) levels in the medium of PBMCs. Neutralization of IFNgamma and IFNgamma receptor using specific antibodies blocked LPS-induced CD40 expression by 44% and 37%, respectively. In summary, LPS-induced CD40 expression on human PBMCs through activation of NFkappaB and JNK, and partially through the induction of IFNgamma production.

• Epidermal growth factor receptor activation by diesel particles is mediated by tyrosine phosphatase inhibition.

  Authors: Tamara L Tal, Philip A Bromberg, Yumee Kim, James M Samet

  Toxicology and applied pharmacology. 10/2008;

  Exposure to particulate matter (PM) is associated with increased cardiopulmonary morbidity and mortality. Diesel exhaust particles (DEP) are a major component of ambient PM and may contribute toExposure to particulate matter (PM) is associated with increased cardiopulmonary morbidity and mortality. Diesel exhaust particles (DEP) are a major component of ambient PM and may contribute to PM-induced pulmonary inflammation. Proinflammatory signaling is mediated by phosphorylation-dependent signaling pathways whose activation is opposed by the activity of protein tyrosine phosphatases (PTPases) which thereby function to maintain signaling quiescence. PTPases contain an invariant catalytic cysteine that is susceptible to electrophilic attack. DEP contain electrophilic oxy-organic compounds that may contribute to the oxidant effects of PM. Therefore, we hypothesized that exposure to DEP impairs PTPase activity allowing for unopposed basal kinase activity. Here we report that exposure to 30 mug/cm(2) DEP for 4 h induces differential activation of signaling in primary cultures of human airway epithelial cells (HAEC), a primary target cell in PM inhalation. In-gel kinase activity assay of HAEC exposed to DEPs of low (L-DEP), intermediate (I-DEP) or high (H-DEP) organic content showed differential activation of intracellular kinases. Exposure to these DEP also induced varying levels of phosphorylation of the receptor tyrosine kinase EGFR in a manner that requires EGFR kinase activity but does not involve receptor dimerization. We demonstrate that treatment with DEP results in an impairment of total and EGFR-directed PTPase activity in HAEC with a potency that correlates with the organic content of these particles. These data show that DEP-induced EGFR-phosphorylation in HAEC is the result of a loss of PTPase activities which normally function to dephosphorylate EGFR in opposition to baseline EGFR kinase activity.

• Regulation of cyclooxygenase-2 expression by cAMP response element and mRNA stability in a human airway epithelial cell line exposed to zinc.

  Authors: Weidong Wu, Robert A Silbajoris, Dongsun Cao, Philip A Bromberg, Qiao Zhang, David B Peden, James M Samet

  Toxicology and applied pharmacology. 05/2008;

  Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are importantExposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are important modulators of airway inflammation. In this study, we characterized the transcriptional and posttranscriptional events that regulate COX-2 expression in a human bronchial epithelial cell line BEAS-2B exposed to Zn(2+). Zn(2+) exposure resulted in pronounced increases in COX-2 mRNA and protein expression, which were prevented by pretreatment with the transcription inhibitor actinomycin D, implying the involvement of transcriptional regulation. This was supported by the observation of increased COX-2 promoter activity in Zn(2+)-treated BEAS-2B cells. Mutation of the cAMP response element (CRE), but not the kappaB-binding sites in the COX-2 promoter markedly reduced COX-2 promoter activity induced by Zn(2+). Inhibition of NFkappaB activation did not block Zn(2+)-induced COX-2 expression. Measurement of mRNA stability demonstrated that Zn(2+) exposure impaired the degradation of COX-2 mRNA in BEAS-2B cells. This message stabilization effect of Zn(2+) exposure was shown to be dependent on the integrity of the 3'-untranslated region found in the COX-2 transcript. Taken together, these data demonstrate that the CRE and mRNA stability regulates COX-2 expression induced in BEAS-2B cells exposed to extracellular Zn(2+).

• Involvement of mitogen-activated protein kinases and NFkappaB in LPS-induced CD40 expression on human monocytic cells.

  Authors: Weidong Wu, Neil E Alexis, Xian Chen, Philip A Bromberg, David B Peden

  Toxicology and applied pharmacology. 05/2008; 228(2):135-43.

  CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanismsCD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFkappaB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFkappaB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFkappaB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFkappaB activation, and CD40 expression. Moreover, blockage of MAPK and NFkappaB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFkappaB.

• Acute pulmonary function response to ozone in young adults as a function of body mass index.

  Authors: William D Bennett, Milan J Hazucha, Lawrence J Folinsbee, Philip A Bromberg, Grace E Kissling, Stephanie J London

  Inhalation toxicology. 11/2007; 19(14):1147-54.

  Recent studies have shown enhanced responsiveness to ozone in obese mice. Adiposity has not been examined as a possible modulator of ozone response in humans. We therefore examined the relationshipRecent studies have shown enhanced responsiveness to ozone in obese mice. Adiposity has not been examined as a possible modulator of ozone response in humans. We therefore examined the relationship between body mass index and the acute spirometric response to ozone (O(3)) exposure among 197 nonasthmatic young adults (aged 18-35 yr) studied in our human exposure facility from 1992 to 1998. Each subject had been exposed to 0.42 ppm O(3) for 1.5 h with intermittent exercise designed to produce a minute ventilation of 20 L/min/m(2) body surface area (BSA). Spirometry (pulmonary function) was measured pre- and immediately postexposure to determine acute ozone-induced changes. The decrement in forced expiratory volume in 1s (Delta FEV1) as percent of baseline was significantly correlated with BMI, r = -0.16, p = .03, with a slightly stronger correlation in women (n = 75), r = -0.22, p = .05, and no significant correlation in men. BMI had a greater range in women than in men in our study. In women greater ozone-induced decrements were seen in overweight (BMI > 25 kg/m(2)) than in normal weight (BMI 18.5 to 25 kg/m(2)), and in normal weight than in underweight (BMI < 18.5 kg/m(2)) for all spirometric variables considered (p trend <or= .022). Although our population studied was predominantly normal weight, we found that higher body mass index may be a modest risk factor for adverse pulmonary effects associated with ozone exposure, especially for women.

• Ozone enhances markers of innate immunity and antigen presentation on airway monocytes in healthy individuals.

  Authors: John C Lay, Neil E Alexis, Steven R Kleeberger, Robert A S Roubey, Bradford D Harris, Philip A Bromberg, Milan J Hazucha, Robert B Devlin, David B Peden

  The Journal of allergy and clinical immunology. 10/2007; 120(3):719-22.

• COX-2 expression induced by diesel particles involves chromatin modification and degradation of HDAC1.

  Authors: Dongsun Cao, Philip A Bromberg, James M Samet

  American journal of respiratory cell and molecular biology. 08/2007; 37(2):232-9.

  Cyclooxygenase-2 (COX-2) plays an important role in the inflammatory response induced by physiologic and stress stimuli. Exposure to diesel exhaust particulate matter (DEP) has been shown to induceCyclooxygenase-2 (COX-2) plays an important role in the inflammatory response induced by physiologic and stress stimuli. Exposure to diesel exhaust particulate matter (DEP) has been shown to induce pulmonary inflammation and exacerbate asthma and chronic obstructive pulmonary disease. DEP is a potent inducer of inflammatory reponses in human airway epithelial cells. The mechanism through which DEP inhalation induces inflammatory mediator expression is not understood. In this report, we demonstrate that DEP can induce the expression of COX-2 gene in a human bronchial epithelial cell line (BEAS-2B) at both transcriptional and protein levels. The induction of COX-2 gene expression involves chromatin modification, in particular acetylation and deacetylation of histones. We show that exposure to DEP increases the acetylation of histone H4 associated with the COX-2 promoter and causes degradation of histone deacetylase 1 (HDAC1). Further, we establish that HDAC1 plays a pivotal role in mediating the transcriptional activation of the COX-2 gene in BEAS-2B cells exposed to DEP, supported by evidence that the down-regulation of HDAC1 using siRNA leads to activation of COX-2 gene expression, whereas overexpression of HDAC1 results in its repression. Finally, DEP exposure induced recruitment of histone acetyltransferase (HAT) p300 to the promoter of the COX-2 gene, suggesting that acetylation is also important in regulating its expression in response to DEP exposure. These results show for the first time acetylation via selective degradation of HDAC1, and that recruitment of HAT plays an important role in DEP-induced expression of the COX-2 gene.