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As nanoparticles (NPs) are cleared via phagocytes of the mononuclear phagocyte system (MPS), we hypothesized that the function of circulating monocytes and dendritic cells (MO/DC) in blood can predict NP clearance (CL). We measured MO/DC phagocytosis and reactive oxygen species (ROS) production in mice, rats, dogs, and patients with refractory solid tumors. Pharmacokinetic studies of polyethylene glycol (PEG)-encapsulated liposomal doxorubicin (PEGylated liposomal doxirubicin [PLD]), CKD-602 (S-CKD602), and cisplatin (SPI-077) were performed at the maximum tolerated dose. MO/DC function was also evaluated in patients with recurrent epithelial ovarian cancer (EOC) administered PLD. Across species, a positive association was observed between cell function and CL of PEGylated liposomes. In patients with EOC, associations were observed between PLD CL and phagocytosis (R(2) = 0.43, P = 0.04) and ROS production (R(2) = 0.61, P = 0.008) in blood MO/DC. These findings suggest that probes of MPS function may help predict PEGylated liposome CL across species and PLD CL in patients with EOC.

Epidemiologic studies suggest that dietary vitamin E is an important candidate intervention for asthma. Our group has shown that daily consumption of vitamin E (γ-tocopherol, γT) has anti-inflammatory actions in both rodent and human phase I studies. The objective of this study was to test whether γT supplementation could mitigate a model of neutrophilic airway inflammation in rats and in healthy human volunteers. F344/N rats were randomized to oral gavage with γT versus placebo, followed by intranasal LPS (20μg) challenge. Bronchoalveolar lavage fluid and lung histology were used to assess airway neutrophil recruitment. In a phase IIa clinical study, 13 nonasthmatic subjects completed a double-blinded, placebo-controlled crossover study in which they consumed either a γT-enriched capsule or a sunflower oil placebo capsule. After 7 days of daily supplementation, they underwent an inhaled LPS challenge. Induced sputum was assessed for neutrophils 6 h after inhaled LPS. The effect of γT compared to placebo on airway neutrophils post-LPS was compared using a repeated-measures analysis of variance. In rats, oral γT supplementation significantly reduced tissue infiltration (p0.05) and accumulation of airway neutrophils (p0.05) that are elicited by intranasal LPS challenge compared to control rats. In human volunteers, γT treatment significantly decreased induced sputum neutrophils (p=0.03) compared to placebo. Oral supplementation with γT reduced airway neutrophil recruitment in both rat and human models of inhaled LPS challenge. These results suggest that γT is a potential therapeutic candidate for prevention or treatment of neutrophilic airway inflammation in diseased populations.

Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate γ-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6) and mild house dust mite-sensitive allergic asthmatics (n = 6) were treated ex vivo with GT (300 µM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p < 0.05) internalization of attached zymosan bioparticles and decreased (p < 0.05) macrophage expression of CD206, CD36 and CD86 in allergic asthmatics but not in controls. Overall, GT caused downregulation of both innate and adaptive immune response elements, and atopic status appears to be an important factor.

Ozone is a commonly encountered environmental air pollutant. In epidemiologic investigations, exposure to increased levels of ambient air O3 has been associated with exacerbations of asthma, chronic obstructive pulmonary disease (COPD), and pneumonia, generally 24–48 hr after exposure occurs (Bernstein et al. 2004; Peden 2001). Controlled chamber exposures to O3 cause an influx of neutrophils to the airway and a decrease in lung function, although these two effects do not correlate with each other, indicating that separate mechanisms account for these effects (Bernstein et al. 2004). O3 exposure also causes increased responsiveness to allergen in allergic asthmatics (Peden 2001). We have recently observed that O3 exposure can also result in increased expression of CD11b, CD14, CD16, CD80, CD86, and HLA-DR on airway dendritic cells (DCs), monocytes, and macrophages (Alexis et al. 2004b). It has been suggested that the action of O3 on airway neutrophils, monocytes, and macrophages accounts for much of the disease outcomes associated with O3 exposure. These inflammatory events also mimic the type of inflammation that occurs with acute viral and bacterial infection and exacerbations of asthma and COPD (Maneechotesuwan et al. 2007; Pauwels 2004). Together, these observations suggest that O3 challenge may be a useful controlled human disease model for screening novel anti-inflammatory pharmaceutical agents in phase I proof-of-concept trials. Holz et al. (2005) tested the utility of a 0.25-ppm O3 challenge as a drug efficacy screen, using a single pre-treatment dose of the established anti-inflammatory agents fluticasone propionate (FP) and oral prednisolone as test agents in a randomized three-arm crossover study in 18 healthy subjects comparing the effect of these two treatments with that of placebo on O3-induced airway inflammation. Holz et al. (2005) reported that, compared with placebo, pretreatment with 2 mg inhaled FP and 50 mg oral prednisolone resulted in a significant reduction in post-O3 sputum neutrophils per milliliter (by 62% and 64%, respectively) and myeloperoxidase (MPO; by 55% and 42%, respectively). These results demonstrated that corticosteroids do inhibit the proinflammatory actions of O3. In the present study, we sought to extend these observations by comparing the effect of a single administration of a high dose of inhaled FP (2 mg) with a dose that is employed in clinical practice for asthma and COPD (0.5 mg) and placebo. Given the importance that monocytes, macrophages, and DCs likely have in the pathophysiology of O3-induced exacerbations of disease, we also examined the effect of these treatments on expression of CD11b/CR3, mCD14, CD16/FcγRIII, CD64/FcγRI, CD86/B7, and HLA-DR on monocytes, macrophages, and DCs recovered from airway sputum. Clara cell protein 16 (CCP16) and surfactant protein D (SP-D) are innate immune molecules and products of airway epithelial cells (Haczku 2006) that can be released to the circulation during lung injury (Holz et al. 2005). CCP16 is induced in the serum of subjects exposed to O3 challenge (Blomberg et al. 2003). We have previously shown that SP-D levels in the lung are significantly altered after O3 inhalation in mice (Kierstein et al. 2006), but whether similar changes can be detected in the human serum is not known. Thus, we evaluated CCP16 and SP-D for their potential utility as serum biomarkers for assessing the effects of inhaled corticosteroids on O3 injury in the respiratory tract.

Background Environmental exposure to endotoxin is a known cause of exacerbation of asthma. Inhaled endotoxin protocols have been used to evaluate airway cell surface phenotypes associated with antigen presentation and innate immunity in healthy volunteers, but not in allergic volunteers. Objectives To establish the safety of challenge with low-dose endotoxin (10,000 endotoxin units) (lipopolysaccharide [LPS]) inhalation in allergic individuals, to measure airway cell surface phenotypes associated with antigen presentation and innate immunity in induced sputum (IS) after LPS challenge, and to conduct gene expression profiling in IS cells to determine which host genetic networks are modified by LPS inhalation. Methods Induced sputum was obtained before and 6 hours after LPS inhalation in 10 allergic volunteers (8 with asthma and 2 with rhinitis). Flow cytometry was used to examine cell surface phenotypes on IS cells. Genomic expression was analyzed on a subset of IS samples (n = 10) using microarray and ingenuity pathway analysis. Results A total of 10,000 endotoxin units of LPS induced significant up-regulation of membrane CD14, CD11b, CD16, HLA-DR, CD86, and Fcɛ receptor 1 on sputum phagocytes and increased expression of genes that influence antigen-presenting surface molecules (HLA-DR, chemokine ligand 2 or monocyte chemoattractant protein 1, v-rel reticuloendotheliosis viral oncogene homolog, prostaglandin-endoperoxide synthase 2 or cyclooxygenase 2, and transforming growth factor β), immune activation (CD14, interleukin 1β, and regulated upon activation, normal T cell expressed and secreted), and inflammation (intracellular adhesion molecule 1 and inhibitory κBα). Gene profiles for nuclear factor κB, interleukin 1, and tumor necrosis factor pathways were also significantly affected. Conclusions Low-dose inhaled endotoxin challenge is safe in allergic individuals with mild to moderate disease. It enhances airway cell surface phenotypes and expression of genes associated with antigen presentation, innate immunity, and inflammation. Microarray with ingenuity pathway analysis can be successfully applied to sputum cells to characterize genetic responses to inhaled exacerbants.

Background We previously reported that inhalation of 5 μg of endotoxin (30,000 endotoxin units [EU]) induced airway neutrophilia and decreased phagocytosis by airway monocytes, macrophages, and neutrophils. Conversely, we recently reported that very low doses of endotoxin, which are not associated with neutrophil influx, enhance response to allergen in the nasal and bronchial airway. Objective We sought to determine whether endotoxin (0-10,000 EU) at doses that do not induce airway neutrophilia prime airway phagocyte function, alter expression of relevant cell-surface receptors (membrane-bound CD14 [mCD14] and CD11b/CR3), and cause induction of a T H 2 cytokine profile in the airway. Methods Thirteen nonallergic healthy volunteers were challenged on separate occasions with escalating doses of Clinical Center Reference Endotoxin (CCRE; 0, 2500, 5000, and 10,000 EU), with 9 volunteers completing the entire dose range. Sputum cells and fluid-phase components were recovered 6 hours after challenge. Sputum inflammatory cells were analyzed by means of flow cytometry for mCD14 and CD11b expression and immune function (phagocytosis of IgG-opsonized zymosan particles). Results At all doses of CCRE, there was no increase in airway neutrophils relative to that caused by saline. However, inhalation of 10,000 EU enhanced phagocytosis (monocytes and macrophages), upregulated expression of CD11b and mCD14 (monocytes and neutrophils), and increased IL-13 levels, whereas IFN-γ levels were significantly decreased. Conclusion The 10,000-EU dose of CCRE is subthreshold for inducing airway neutrophilia but primes phagocyte function and cell-surface receptor expression in the presence of increased IL-13 and decreased IFN-γ levels. We speculate that low-dose endotoxin challenge skews airway inflammation in a T H 2 direction in vivo .

Objective To determine if the GSTM1 null genotype is a risk factor for increased inflammatory response to inhaled endotoxin. Methods 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. Results 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. Conclusion 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.

Epidemiologic studies suggest that dietary vitamin E is a candidate intervention for atopic disease. We used in vitro and ex vivo exposures to test the hypothesis that the most common dietary isoform of vitamin E, γ-tocopherol (γT), could suppress FceRI-mediated basophil activation. Rat basophilic leukemia (RBL)-SX38 cells that express human FceRI were treated with or without γT, followed by stimulation with α-IgE. In the ex vivo study, 20 Der f 1-allergic volunteers consumed a γT-enriched supplement for 7 days. Their basophils were challenged ex vivo with α-IgE and graded doses of Der f 1 before and after the supplementation period. γt treatment of RBL-SX38 cells significantly reduced basophil degranulation and de novo TH2 cytokine production. Daily consumption of a γT-rich supplement by dust mite-allergic volunteers reduced basophil activation after ex vivo dust mite challenge. Vitamin E supplements rich in γT may be useful adjuncts in decreasing atopic disease.

Pulmonary inflammation secondary to oxidant generation catalyzed by transition metals associated with inhaled particles is one factor postulated to underlie the acute health effects of particulate air pollution. We postulated that inhaled iron oxide particles with associated amounts of soluble iron should induce mild pulmonary inflammation and lead to altered alveolar epithelial integrity and altered gas exchange. To test this hypothesis we examined the effects of inhaled iron oxide particles on alveolar epithelial permeability. Sixteen healthy subjects inhaled aerosols of iron oxide particles (1.5 microm mass median aerodynamic diameter) having either high or low water-soluble iron content [3.26 +/- 0.25 (SE) and 0.14 +/- 0.04 microg soluble iron/mg of particles, respectively] for 30 min at an average mass concentration of 12.7 mg/m(3). Alveolar epithelial permeability was assessed by measuring the pulmonary clearance of an inhaled radiolabeled tracer molecule ((99m)Tc-DTPA, diethylene triamine pentaacetic acid) using a gamma camera at 1/2 h and 24 h post particle exposure. Carbon monoxide lung diffusing capacity (DL(CO)) and spirometry were also performed before and after breathing the iron oxide. As a control, on a separate day, the procedures were duplicated except that the subject breathed particle-free air. For those subjects breathing aerosols with high soluble iron, we found no significant difference in DTPA clearance half-times after breathing particles versus particle-free air either at 1/2 h (97.4 +/- 15.4 vs. 116.1 +/- 15.5 min, respectively) or 24 h postinhalation (105.1 +/- 13.8 vs. 106.9 +/- 12.9 min, respectively). Likewise, for those subjects breathing aerosols with low soluble iron content we found no significant difference in DTPA clearance half-times after breathing particles versus particle-free air either at 1/2 h (108.6 +/- 31.9 vs. 95.6 +/- 10.8 min, respectively) or 24 h postinhalation (130.0 +/- 18.0 vs. 105.8 +/- 13.7 min, respectively). We found no significant differences in DL(CO) between particle exposures and air exposures. Minor differences in spirometric measurements were noted but were not statistically significant. We conclude that inhalation of iron oxide particles did not cause an appreciable alteration of alveolar epithelial permeability, lung diffusing capacity, or pulmonary function in healthy subjects under the studied conditions.

Bronchoalveolar lavage (BAL) was used to sample lung cells and biochemical components in the lung air spaces at various times from 1 to 91 d after intrapulmonary instillation of 2.6 microm-diameter iron oxide particles in human subjects. The instillation of particles induced transient acute inflammation during the first day post instillation (PI), characterized by increased numbers of neutrophils and alveolar macrophages as well as increased amounts of protein, lactate dehydrogenase, and interleukin-8 in BAL fluids. This response was subclinical and was resolved within 4 d PI. A similar dose-dependent response was seen in rats 1 d after intratracheal instillation of the same particles. The particles contained small amounts of soluble iron (240 ng/mg) and possessed the capacity to catalyze oxidant generation in vitro. Our findings indicate that the acute inflammation after particle exposure may, at least partially, be the result of oxidant generation catalyzed by the presence of residual amounts of ferric ion, ferric hydroxides, or oxyhydroxides associated with the particles. These findings may have relevance to the acute health effects associated with increased levels of ambient particulate air pollutants.

The atmosphere constitutes a prime vehicle for the movement and redistribution of metals. Metal exposure can be associated with an oxidative stress. We tested the hypothesis that, in response to an iron-containing particle, the human respiratory tract will demonstrate an increased expression of both lactoferrin and ferritin as the host attempts to transport and store the metal in a chemically less-reactive form and therefore diminish the oxidative stress the particle presents. Subjects ( n = 22) were instilled with 20 ml of saline and 20 ml of an iron-containing particle suspended in saline in a right middle lobe bronchus and a lingular bronchus, respectively. At either 1, 2, or 4 days after this exposure, the volunteer was lavaged for a sample of the lower respiratory tract, and concentrations ofl-ferritin, transferrin, and lactoferrin were measured by enzyme immunoassay, immunoprecipitin analysis, and enzyme-linked immunosorbent assay (ELISA), respectively. Transferrin receptor was also quantified by ELISA. The concentrations of l-ferritin in the lavage fluid of lung exposed to particles were significantly increased relative to the levels of the protein in the segment exposed to saline. Relative to saline instillation, transferrin was significantly diminished after exposure to the iron-containing particle, whereas both lactoferrin and transferrin receptor concentrations in the segment of the lung exposed to the particle were significantly elevated. We conclude that instillation of an iron-containing particle was associated with a disequilibrium in iron metabolism in the lower respiratory tract. The response included increased ferritin and lactoferrin concentrations, whereas transferrin concentrations diminished. This coordinated series of reactions by the host effects a decrease in the availability of catalytically reactive iron to likely diminish the consequent oxidative stress to the human host.

Background Exposure to ozone activates innate immune function and causes neutrophilic (PMN) airway inflammation that in some individuals is robustly elevated. The interplay between immuno-inflammatory function and genomic signaling in those with heightened inflammatory responsiveness to ozone is not well understood. Objectives Determine baseline predictors and post exposure discriminators for the immuno-inflammatory response to ozone in inflammatory responsive adult volunteers. Methods Sputum induction was performed on 27 individuals before and after a two hour chamber exposure to 0.4 ppm ozone. Subjects were classified as inflammatory responders or non-responders to ozone based on their PMN response. Innate immune function, inflammatory cell and cytokine modulation and transcriptional signaling pathways were measured in sputum. Results Post exposure, responders showed activated innate immune function (CD16: 31,004 MFI vs 8988 MFI; CD11b: 44,986 MFI vs 24,770 MFI; CD80: 2236 MFI vs 1506 MFI; IL-8: 37,603 pg/ml vs 2828 pg/ml; and IL-1β: 1380 pg/ml vs 318 pg/ml) with muted signaling of immune cell trafficking pathways. In contrast, non-responders displayed decreased innate immune activity (CD16, CD80; phagocytosis: 2 particles/PMN vs 4 particles/PMN) post exposure that was accompanied by a heightened signaling of immune cell trafficking pathways. Conclusions Inflammatory responsive and non responsive individuals to ozone show an inverse relationship between immune cell trafficking and immuno-inflammatory functional responses to ozone. These distinct genomic signatures may further our understanding about ozone-induced morbidity in individuals with different levels of inflammatory responsiveness.

The evaluation of sputum leukocytes by flow cytometry (FCM) is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris.To develop a gating strategy based on specific antibody panels in combination with light scatter properties for flow cytometric evaluation of sputum cells.Healthy and mild asthmatic volunteers underwent sputum induction. Manually selected mucus "plug" material was treated with dithiothreitol, filtered and total leukocytes acquired. Multicolor FCM was performed using specific gating strategies based on light scatter properties, differential expression of CD45 and cell lineage markers to discriminate leukocytes from squamous epithelial cells and debris.The combination of forward scatter and CD45 expression reliably segregated sputum leukocytes from contaminating squamous epithelial cells and debris. Overlap of major leukocyte populations (neutrophils, macrophages/monocytes) required the use of specific antibodies (e.g. CD16, CD64, CD14, HLA-DR) that differentiated granulocytes from monocytes and macrophages. These gating strategies allowed identification of small populations of eosinophils, CD11c+ myeloid dendritic cells, B-cells and natural killer cells.Multicolor FCM can be successfully applied to sputum samples to identify and characterize leukocyte populations residing on the surfaces of the central airways.This research describes detailed methods to overcome difficulties associated with FCM of sputum samples, which previously has been lacking in the literature. FCM of sputum samples can provide valuable information on inflammation and immunological response elements in the bronchial airways for both clinical diagnostic and research applications and can be a useful tool in inhalation toxicology for assessing health effects of inhaled environmental pollutants.

Exposure to ozone causes a decrease in spirometric lung function and an increase in airway inflammation in healthy young adults at concentrations as low as 0.08 ppm, close to the National Ambient Air Quality Standard for ground level ozone.To test whether airway effects occur below the current ozone standard and if they are more pronounced in potentially susceptible individuals, such as those deficient in the antioxidant gene glutathione S-transferase mu 1 (GSTM1).Pulmonary function and subjective symptoms were measured in 59 healthy young adults (19-35 yr) immediately before and after exposure to 0.0 (clean air, CA) and 0.06 ppm ozone for 6.6 hours in a chamber while undergoing intermittent moderate exercise. The polymorphonuclear neutrophil (PMN) influx was measured in 24 subjects 16 to 18 hours postexposure.Subjects experienced a significantly greater (P = 0.008) change in FEV(1) (± SE) immediately after exposure to 0.06 ppm ozone compared with CA (-1.71 ± 0.50% vs. -0.002 ± 0.46%). The decrement in FVC was also greater (P = 0.02) after ozone versus CA (-2.32 ± 0.41% vs. -1.13 ± 0.34%). Similarly, changes in %PMN were greater after ozone (54.0 ± 4.6%) than CA (38.3 ± 3.7%) exposure (P0.001). Symptom scores were not different between ozone versus CA. There were no significant differences in changes in FEV(1), FVC, and %PMN between subjects with GSTM1-positive and GSTM1-null genotypes.Exposure of healthy young adults to 0.06 ppm ozone for 6.6 hours causes a significant decrement of FEV(1) and an increase in neutrophilic inflammation in the airways. GSTM1 genotype alone appears to have no significant role in modifying the effects.

Recent studies have suggested considerable particle retention in the bronchial, ciliated airways 24 hours after depositing in the lung. A significant presence of particles in the ciliated ...

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 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.

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 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.

The uptake of inhaled particulate matter by airway phagocytes is an important defence mechanism contributing to the clearance of potentially toxic substances, including aeroallergens, from the lung. Since airway monocytes and macrophages can also function as antigen presenting cells, their ability to engulf materials deposited on the airway surface is of particular interest in patients with allergic asthma. To determine whether airway mononuclear phagocytes of patients with allergic asthma might have enhanced phagocytic activity, the in vivo uptake of inhaled radiolabelled particles was compared in 10 patients with mild allergic asthma and 8 healthy (non-allergic) individuals.Phagocyte function was assessed by quantifying the proportion of radioactivity associated with cellular and supernatant fractions of induced sputum 2 h after inhalation of radiolabelled sulfur colloid particles. All subjects were pretreated with albuterol before sputum induction. A standardised breathing pattern was used to target aerosol deposition in the bronchial airways.In vivo particle uptake by airway cells was significantly greater in patients with asthma than in healthy volunteers (57.2% (95% CI 46.5% to 67.9%) vs 22.3% (95% CI 4.9% to 39.6%), p0.01), as was in vitro phagocytosis of opsonised zymosan-A bioparticles. There was also a significant correlation (r = 0.85, p0.01) between the percentage of sputum mononuclear phagocytes and the percentage uptake of particles in the patients with asthma but not in the control subjects.In vivo particle uptake by airway macrophages is enhanced in persons with mild asthma. Enhanced uptake and processing of particulate antigens could contribute to the pathogenesis and progression of allergic airways disease and may contribute to the increased risk of disease exacerbation associated with particulate exposure.

We combined two techniques, radiolabeled aerosol inhalation delivery and induced sputum, to examine in vivo the time course of particle uptake by airway macrophages in 10 healthy volunteers. On three separate visits, induced sputum was obtained 40, 100, and 160 min after inhalation of radiolabeled sulfur colloid (SC) aerosol (Tc99 m-SC, 0.2 microm colloid size delivered in 6-microm droplets). On a fourth visit (control) with no SC inhalation, induced sputum was obtained and SC particles were incubated (37 degrees C) in vitro with sputum cells for 40, 100, and 160 min (matching the times associated with in vivo sampling). Total and differential cell counts were recorded for each sputum sample. Compared with 40 min (6 +/- 3%), uptake in vivo was significantly elevated at 100 (31 +/- 5%) and 160 min (27 +/- 4%); both were strongly associated with the number of airway macrophages (R = 0.8 and 0.7, respectively); and the number and proportion of macrophages at 40 min were significantly (P < 0.05) elevated compared with control (1,248 +/- 256 versus 555 +/- 114 cells/mg; 76 +/- 6% versus 60 +/- 5%). Uptake in vitro increased in a linear fashion over time and was maximal at 160 min (40 min, 12 +/- 2%; 100 min, 16 +/- 4%; 160 min, 24 +/- 6%). These data suggest that airway surface macrophages in healthy subjects rapidly engulf insoluble particles. Further, macrophage recruitment and phagocytosis-modifying agents are factors in vivo that likely affect particle uptake and its time course.

Background In vitro , endotoxin on coarse fraction particulate matter (PM 2.5-10 ) accounts for the majority of the ability of PM 2.5-10 to induce cytokine responses from alveolar macrophages. Objective We examined in vivo whether inhaled PM 2.5-10 from local ambient air induce inflammatory and immune responses in the airways of healthy human beings and whether biologic material on PM 2.5-10 accounts for these effects. Methods On 3 separate visits, 9 healthy subjects inhaled nebulized saline (0.9%, control), PM 2.5-10 collected from local ambient air that was heated to inactivate biological material (PM 2.5-10 − ), or nonheated PM (PM 2.5-10 + ). PM 2.5-10 deposition (∼0.65 mg/subject) targeted the bronchial airways (confirmed by using radiolabeled aerosol), and induced sputum was obtained 2 to 3 hours postinhalation for analysis of cellular and biochemical markers of inflammation and innate immune function. Results Inhaled PM 2.5-10 + induced elevated inflammation (% PMNs, macrophage mRNA TNF-α), increased eotaxin, upregulated immune surface phenotypes on macrophages (mCD14, CD11b, HLA-DR), and increased phagocytosis (monocytes) versus saline ( P 2.5-10 (PM 2.5-10 − ) had no effect on neutrophilia but significantly ( P .05) attenuated mRNA TNF-α, eotaxin levels, cell surface marker responses, and phagocytosis. Conclusion Biological components of PM 2.5-10 are not necessary to induce neutrophil responses but are essential in mediating macrophage responses. The ability of PM 2.5-10 to activate monocytic cells and potentially skew the airways toward an allergic phenotype by enhancing eotaxin levels may enhance responses to allergens or bacteria in individuals with allergy. Clinical implications PM 2.5-10 might enhance the response of individuals with allergy to airborne bacteria.

We have been studying the innate immune response of airways cells of healthy human volunteers to inhaled LPS, a Toll-like receptor 4 (TLR4) ligand, and have shown that macrophage phagocytic capacity is blunted.Because a primary feature of dendritic cell (DC) maturation is a loss of phagocytic capacity, we sought to determine whether acute LPS inhalation in healthy volunteers promotes DC maturation in vivo.Phagocytosis (IgG-opsonized zymosan particles) and cell-surface phenotypes were analyzed by flow cytometry of induced sputum cells obtained before and 6 hours after Clinical Center Reference Endotoxin (CCRE; 20,000 EU) inhalation in 9 healthy volunteers.Neutrophils were elevated in the airways after CCRE inhalation (67% +/- 6% vs 37% +/- 6%; P.05). Phagocytosis (monocytes, macrophages) was blunted (73%, 46%; P.05) and negatively correlated with PMN influx ( R = -0.73; P.05) after CCRE inhalation. GM-CSF and IL-1beta, potent DC maturation agents, were elevated after versus before CCRE inhalation (217 pg/mL +/- 103 pg/mL vs 722 pg/mL +/- 202 pg/mL; 83 pg/mL +/- 24 pg/mL vs 148 pg/mL +/- 37 pg/mL, respectively; P.05). Markers of DC maturation (CD80, CD86, HLA-DR) were upregulated on monocytes and macrophages ( P.05), and discrete populations of mature DC were observed ( P.05) after CCRE inhalation.Inhaled LPS, directly through TLR4 stimulation of immature DC and/or indirectly through stimulation of GM-CSF and IL-1beta, induces pulmonary DC maturation in vivo . Inhaled LPS may enhance allergic airways responses to air pollution through its ability to induce DC maturation.

We used a gamma camera to monitor the retention and clearance of radiolabeled human serum albumin (HSA), a water-soluble material with molecular weight of 66,000 Daltons, and radiolabeled sulfur colloid (SC), an insoluble submicron (0.22 microm) particle, following localized deposition in a 4-5-mm bronchus in each of five dogs. The average retention time of HSA was significantly greater than that of SC both in the deposition site (23.3 +/- 2.3 vs. 18.2 +/- 2.9 min respectively, p0.05) and in the clearance pathway (26.5 +/- 2.0 vs. 22.0 +/- 1.4 min, respectively, p0.05). The mean percent retention at the deposition site at 60 minutes post-deposition also was significantly greater for HSA than for SC (33.5 +/- 10.1 vs. 11.6 +/- 4.7%, respectively, p0.05). The percentage of HSA which had cleared to the level of the cuffed endotracheal tube was significantly less than that of SC (18.0 +/- 6.7 vs. 35.8 +/- 3.5%, respectively, p0.05) at 60 min post-deposition. These findings indicate that a low-permeating water-soluble material such as HSA deposited on the surface of an airway remains in contact with the sensitive airway epithelium to a greater extent than does a solid insoluble particle. Based on our results, we speculate that the slower clearance of HSA compared to SC was likely due to diffusion of a greater portion of the HSA into the periciliary sol layer which may be transported less efficiently than the mucus layer during mucociliary clearance. Additionally, some degree of uptake of HSA by bronchial epithelium may have contributed to its increased retention.

Bronchoalveolar lavage (BAL) was used to sample retention of particles within the alveolar macrophage (AM) compartment at various times from 1 to 91 d following intrapulmonary instillation of 2. 6-microm-diameter iron oxide (Fe2O3) particles in human subjects. Particles were cleared from the lavagable AM compartment in a biphasic pattern, with a rapid-phase clearance half-time of 0.5 d and long-term clearance half-time of 110 d, comparable to retention kinetics determined by more traditional methods. The intracellular distribution of particles within lavaged AMs was similar in bronchial and alveolar BAL fractions. AMs with high intracellular particle burdens disappeared from the lavagable phagocytic AM population disproportionately more rapidly (shorter clearance half-time) than did AMs with lower particle burdens, consistent with the occurrence of a particle redistribution phenomenon as previously described in similar studies in rats. The rates of AM disappearance from the various particle burden categories was generally slightly slower in bronchial fractions than in alveolar fractions. The instillation of particles induced a transient acute inflammatory response at 24 h postinstillation (PI), characterized by increased numbers of neutrophils and alveolar macrophages in BAL fluids. This response was subclinical and was resolved within 4 d PI.

Recent studies have challenged the generally accepted hypothesis that bronchial particle clearance is complete within 24–48 h postdeposition. We studied bronchial retention of inert particles using a bronchoscope and microspray nozzle to localize deposition in a bronchus while avoiding alveolar deposition. Six-microliter aliquots (444 kBq) of submicrometer (number mean diameter = 0.22 microns, geometric standard deviation = 1.75) technetium-99m-labeled (99mTc) sulfur colloid (SC) particles (n = 6) or the unbound radiolabel 99mTc-pertechnetate (99mTcO4-; n = 3) were sprayed onto a 5-mm-diam bronchus in halothane-anesthetized dogs. Radioactivity at the deposition site and clearance pathway was monitored externally with a gamma camera beginning immediately postspray. Bronchial retention of SC was 8.5 +/- 2.4 and 1.5 +/- 0.7% at 3 and 24 h postspray, respectively. Tracheal mucus velocity was measured at 10.4 +/- 2.2 mm/min. For comparison, clearance of inhaled submicrometer SC particles was also measured in the same dogs. Retention of inhaled aerosolized SC (peripheral lung deposition) was 98.1 +/- 1.1 and 76.3 +/- 1.8% at 3 and 24 h, respectively. 99mTcO4- cleared from the bronchi slightly more rapidly than did SC. Radioactivity was readily detected in the blood after deposition of 99mTcO4- but not of SC. Thus SC cleared by mucociliary transport, whereas 99mTcO4- cleared predominantly by transepithelial absorption. We conclude that clearance of submicrometer particles from a 5-mm conducting airway is very nearly complete by 24 h, with approximately 92% of the clearance occurring within the first 3 h postdeposition.

Background Atopic asthmatic patients are reported to be more sensitive to the effects of environmental endotoxin (LPS) than healthy volunteers (HVs). It is unknown whether this sensitivity is due to dysregulated inflammatory responses after LPS exposure in atopic asthmatic patients. Objective We sought to test the hypothesis that atopic asthmatic patients respond differentially to inhaled LPS challenge compared with HVs. Methods Thirteen allergic asthmatic (AA) patients and 18 nonallergic nonasthmatic subjects (healthy volunteers [HVs]) underwent an inhalation challenge to 20,000 endotoxin units of Clinical Center Reference Endotoxin (LPS). Induced sputum and peripheral blood were obtained at baseline and 6 hours after inhaled LPS challenge. Sputum and blood samples were assayed for changes in inflammatory cell numbers and cytokine and cell-surface marker levels on monocytes and macrophages. Results The percentage of neutrophils in sputum (%PMN) in induced sputum similarly and significantly increased in both HVs and AA patients after inhaled LPS challenge. However, the absolute numbers of leukocytes and PMNs recruited to the airways were significantly lower in AA patients compared with those seen in HVs with inhaled LPS challenge. Sputum levels of IL-6 and TNF-α were significantly increased in both cohorts, but levels of IL-1β and IL-18 were only significantly increased in the HV group. Cell-surface expression of Toll-like receptors 4 and 2 were significantly enhanced only in the HV group. Conclusions The airway inflammatory response to inhaled LPS challenge is blunted in AA patients compared with that seen in HVs and accompanied by reductions in airway neutrophilia and inflammasome-dependent cytokine production. These factors might contribute to increased susceptibility to airway microbial infection or colonization in AA patients.

2591 Background: A phenotypic probe is a test that can be administered to a patient as a potential indicator of a drug’s PK/PD which can then be used to individualize therapy. Since nanoparticles are cleared via the MPS, including blood monocytes (MO), we hypothesize that circulating MO could potentially play a major role in, and be a surrogate marker of nanoparticle clearance (CL). Furthermore, we postulate that the ability to measure MO functional activity in blood samples can be used to predict CL and subsequently PD endpoints such as progression free survival (PFS) and hand-foot syndrome (HFS), in patients (pt). Methods: PLD 30/40 mg/m2 IV x 1 with or without carboplatin (AUC = 5 mg/mL/min) was administered to pts (n=10) with recurrent ovarian cancer. Serial PK samples were obtained at time 0 to day 28 post dose. Plasma was processed to measure encapsulated and released doxorubicin using solid phase separation and HPLC. Data was analyzed with WinNonlin to obtain PK parameters. MO and granulocyte phagocytic function (uptake of FITC-labeled opsonized E. Coli) and oxidative burst activity (intracellular oxidation of dihydrorhodamine 123) were assessed via flow cytometry at 0, 48, 96 h, and on day 28. For both assays, changes in mean fluorescence intensity of the gated MO population, following incubation of whole blood with the appropriate substrate, served as an index of activity. Pts were followed from the first dose of PLD until time of disease progression. Results: There was a direct linear relationship between encapsulated doxorubicin CL and both phagocytosis (R2 = 0.87) and oxidative burst activity (R2 = 0.64) in blood MO. Similarly, phagocytosis (R2 = 0.77) and oxidative burst probes correlated with PFS (R2 = 0.67) in the 4 pts who progressed while on PLD. Oxidative burst also correlated with degree of HFS (R2 = 0.56). There was no relationship between phagocytosis and oxidative burst in granulocytes and PK/PD of PLD. Conclusions: These findings indicate that probes of MPS function predict PLD CL, HFS and PFS and thus may be useful for individualizing PLD therapy in ovarian cancer and other malignancies.

2550 Background: A phenotypic probe is a test that can be administered to a patient as a potential indicator of a drug’s PK which can then be used to individualize therapy. PK of nanoparticle agent...

Background 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 in modulation of ozone-induced inflammation has not been determined. Objective We sought to determine whether atopic status modulates ozone response phenotypes in human subjects. Methods 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. Results 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-1β levels and airway macrophage Toll-like receptor 4, FceRI, 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. Conclusion 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.

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. 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.

The deposition, retention, and clearance of inhaled cobalt oxide particles from the lungs of calves with acute inflammatory lung injury induced by parainfluenza-3 virus (PI-3) were examined. Acute pulmonary inflammation was induced by nebulization with 10(9) TCID50 of PI-3 virus on two successive days, and animals were subsequently exposed to an aerosol of particulate cobalt oxide (geometric mean diameter 0.54-0.65 microns) seven days post-virus infection (dpi). Pulmonary lesions at 7 dpi were typical of PI-3 pneumonitis and were characterized by patchy aveolitis and bronchiolitis with accumulations of neutrophils, macrophages, fibrin, and inflammatory debris. Calves were killed at 0, 7, and 21 days post-aerosol exposure (dpe) to evaluate particle clearance and retention by assay for cobalt in lung tissues, bronchoalveolar washings, and tracheobronchial lymph nodes. Control animals had a typically biphasic clearance pattern with rapid initial clearance of 50% of the initial lung burden (ILB) by 7 dpe followed by slower prolonged clearance. Clearance was significantly retarded (P less than 0.05) in calves with viral-induced acute inflammatory lung injury; 90% of the ILB was retained at 7 dpe. Essentially all particles recoverable by bronchoalveolar lavage were intracellular within pulmonary alveolar macrophages (PAM) in both experimental and control groups, but interstitial sequestration of particles within PAM was commonly observed only in the lungs of calves with viral pneumonitis. Pneumonic calves also exhibited retarded translocation of particles to regional lymph nodes. The results document impaired particulate clearance from acutely inflamed lungs, and implicate decreased mucociliary clearance and interstitial sequestration within PAM as the major contributing factors. These functional alterations would be expected to enhance the progression of virus-induced acute pulmonary inflammatory injury.

Twenty one- to two-month-old Holstein, Angus, or Holstein-Angus crossbred calves were given intravenous complete Freund's adjuvant and their lungs were examined at 24 hours to 30 days post-injection. Two calves given intravenous saline served as normal controls. The evolving pulmonary inflammatory response was characterized initially by multifocal vasculitis and acute multifocal exudative pneumonitis which progressed to a granulomatous interstitial pneumonitis by seven days post-injection. Discrete granulomas characterized the lesions in the lungs and lymph nodes at 30 days post-injection, and granulomas also were seen in liver, kidney, and spleen. Clinically, the calves were tachypneic and pyrexic during the first week post-injection. Four calves developed acute pulmonary edema and died during, or shortly after, administration of the adjuvant. This model of experimental pneumonia in calves is similar to complete Freund's adjuvant-induced experimental pneumonia in other species. It is reproducible and predictable in its course of development and resolution, and provides a useful model for studying basic mechanisms of pulmonary inflammatory injury and repair in the bovine lung.