A Preclinical Study in Rhesus Macaques for Cystic Fibrosis to Assess Gene Transfer and Transduction by AAV1 and AAV5 with a Dual-Luciferase Reporter System.

Cystic fibrosis (CF) is an autosomal recessive disease that is potentially treatable by gene therapy. Since the identification of the gene encoding CF transmembrane conductance regulator, a number of preclinical and clinical trials have been conducted using the first generation of adeno-associated virus, AAV2. All these studies showed that AAV gene therapy for CF is safe, but clinical benefit was not clearly demonstrated. Thus, a new generation of AAV vectors based on other serotypes is needed to move the field forward. This study tested two AAV serotypes (AAV1 and AAV5) using a dual-luciferase reporter system with firefly and Renilla luciferase genes packaged into AAV1 or AAV5, respectively. Two male and two female Rhesus macaques were each instilled in their lungs with both serotypes using a Penn-Century microsprayer. Both AAV1 and AAV5 vector genomes were detected in all the lung samples when measured at the time of necropsy, 45 days after instillation. However, the vector genome number for AAV1 was at least 10-fold higher than for AAV5. Likewise, luciferase activity was also detected in the same samples at 45 days. AAV1-derived activity was not statistically greater than that derived from AAV5. These data suggest that gene transfer is greater for AAV1 than for AAV5 in macaque lungs. Serum neutralizing antibodies were increased dramatically against both serotypes but were less abundant with AAV1 than with AAV5. No adverse events were noted, again indicating that AAV gene therapy is safe. These results suggest that with more lung-tropic serotypes such as AAV1, new clinical studies of gene therapy using AAV are warranted.

Safety and biodistribution assessment of sc-rAAV2.5IL-1Ra administered via intra-articular injection in a mono-iodoacetate-induced osteoarthritis rat model.

Interleukin-1 (IL-1) plays an important role in the pathophysiology of osteoarthritis (OA), and gene transfer of IL-1 receptor antagonist (IL-1Ra) holds promise for OA treatment. A preclinical safety and biodistribution study evaluated a self-complementary adeno-associated viral vector carrying rat IL-1Ra transgene (sc-rAAV2.5rIL-1Ra) at 5 × 10(8), 5 × 10(9), or 5 × 10(10) vg/knee, or human IL-1Ra transgene (sc-rAAV2.5hIL-1Ra) at 5 × 10(10) vg/knee, in Wistar rats with mono-iodoacetate (MIA)-induced OA at days 7, 26, 91, 180, and 364 following intra-articular injection. The MIA-induced OA lesions were consistent with the published data on this model. The vector genomes persisted in the injected knees for up to a year with only limited vector leakage to systemic circulation and uptake in tissues outside the knee. Low levels of IL-1Ra expression and mitigation of OA lesions were observed in the vector-injected knees, albeit inconsistently. Neutralizing antibodies against the vector capsid developed in a dose-dependent manner, but only the human vector induced a small splenic T-cell immune response to the vector capsid. No local or systemic toxicity attributable to vector administration was identified in the rats as indicated by clinical signs, body weight, feed consumption, clinical pathology, and gross and microscopic pathology through day 364. Taken together, the gene therapy vector demonstrated a favorable safety profile.

Evaluation of gene expression changes in human primary lung epithelial cells following 24-hr exposures to inorganic arsenic and its methylated metabolites and to arsenic trioxide.

The concentration response for altered gene expression in primary lung epithelial cells was determined following two treatments with arsenicals: (1) a mixture of trivalent arsenic compounds representative of urinary arsenic concentrations in exposed human populations, and (2) arsenite (As2 O3 ) a common form of inhaled arsenic dust that is frequently used in both in vivo and in vitro experimental exposures. Biochemical assays did not detect any evidence of cytotoxicity at the concentrations used, apart from a concentration-related increase in cellular heme oxygenase that was also indicated by the genomic analysis. Cell signal pathway enrichment analysis indicated similar responses to both treatments, with concentration-related responses in pathways related to cell adhesion, cytoskeleton remodeling, development (morphogenesis), cell cycle control, and to a lesser extent inflammatory responses. These cellular responses to arsenic were consistent with those observed in a previous study with primary uroepithelial cells. Benchmark dose analysis also demonstrated similar potency of the two treatments as well as comparable sensitivity of the two cell types. A number of genes showing similar concentration-dependent expression across individuals in both bladder and lung cells were identified, including heme oxygenase 1, thioredoxin reductase, DNA damage binding protein 2, and thrombomodulin. The data on human primary lung cells from this study, together with the data from human primary uroepithelial cells, support a conclusion that biological responses to arsenic by human cells under study conditions are unlikely to occur at concentrations below 0.1 µM. Environ. Mol. Mutagen. 56:477-490, 2015. © 2015 Wiley Periodicals, Inc.

Part 1. Assessment of carcinogenicity and biologic responses in rats after lifetime inhalation of new-technology diesel exhaust in the ACES bioassay.

The Health Effects Institute and its partners conceived and funded a program to characterize the emissions from heavy-duty diesel engines compliant with the 2007 and 2010 on-road emissions standards in the United States and to evaluate indicators of lung toxicity in rats and mice exposed repeatedly to 2007-compliant new-technology diesel exhaust (NTDE*). The a priori hypothesis of this Advanced Collaborative Emissions Study (ACES) was that 2007-compliant on-road diesel emissions "... will not cause an increase in tumor formation or substantial toxic effects in rats and mice at the highest concentration of exhaust that can be used ... although some biological effects may occur." This hypothesis was tested at the Lovelace Respiratory Research Institute (LRRI) by exposing rats by chronic inhalation as a carcinogenicity bioassay. Indicators of pulmonary toxicity in rats were measured after 1, 3, 12, 24, and 28-30 months of exposure. Similar indicators of pulmonary toxicity were measured in mice, as an interspecies comparison of the effects of subchronic exposure, after 1 and 3 months of exposure. A previous HEI report (Mauderly and McDonald 2012) described the operation of the engine and exposure systems and the characteristics of the exposure atmospheres during system commissioning. Another HEI report described the biologic responses in mice and rats after subchronic exposure to NTDE (McDonald et al. 2012). The primary motivation for the present chronic study was to evaluate the effects of NTDE in rats in the context of previous studies that had shown neoplastic lung lesions in rats exposed chronically to traditional technology diesel exhaust (TDE) (i.e., exhaust from diesel engines built before the 2007 U.S. requirements went into effect). The hypothesis was largely based on the marked reduction of diesel particulate matter (DPM) in NTDE compared with emissions from older diesel engine and fuel technologies, although other emissions were also reduced. The DPM component of TDE was considered the primary driver of lung tumorigenesis in rats exposed chronically to historical diesel emissions. Emissions from a 2007-compliant, 500-horsepower-class engine and after treatment system operated on a variable-duty cycle were used to generate the animal inhalation test atmospheres. Four groups were exposed to one of three concentrations (dilutions) of exhaust combined with crankcase emissions, or to clean air as a negative control. Dilutions of exhaust were set to yield average integrated concentrations of 4.2, 0.8, and 0.1 ppm nitrogen dioxide (NO2). Exposure atmospheres were analyzed by daily measurements of key effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors. components and periodic detailed physical-chemical characterizations. Exposures were conducted 16 hours/day (overnight, during the rats' most active period), 5 days/week. Responses to exposure were evaluated via hematology, serum chemistry, bronchoalveolar lavage (BAL), lung cell proliferation, histopathology, and pulmonary function. The exposures were accomplished as planned, with average integrated exposure concentrations within 20% of the target dilutions. The major components from exhaust were the gaseous inorganic compounds, nitrogen monoxide (NO), NO2, and carbon monoxide (CO). Minor components included low concentrations of DPM and volatile and semi-volatile organic compounds (VOCs and SVOCs). Among the more than 100 biologic response variables evaluated, the majority showed no significant difference from control as a result of exposure to NTDE. The major outcome of this study was the absence of pre-neoplastic lung lesions, primary lung neoplasia, or neoplasia of any type attributable to NTDE exposure. The lung lesions that did occur were minimal to mild, occurred only at the highest exposure level, and were characterized by an increased number and prominence of basophilic epithelial cells (considered reactive or regenerative) lining distal terminal bronchioles, alveolar ducts, and adjacent alveoli (termed in this report "Hyperplasia; Epithelial; Periacinar"), which often had a minimal increase in subjacent fibrous stroma (termed "Fibrosis; Interstitial; Periacinar"). Slight epithelial metaplastic change to a cuboidal morphology, often demonstrating cilia, was also noted in some animals (termed "Bronchiolization"). In addition to the epithelial proliferation, there was occasionally a subtle accumulation of pulmonary alveolar macrophages (termed "Accumulation; Macrophage") in affected areas. The findings in the lung progressed slightly from 3 to 12 months, without further progression between 12 months and the final sacrifice at 28 or 30 months. In addition to the histologic findings, there were biochemical changes in the lung tissue and lavage fluid that indicated mild inflammation and oxidative stress. Generally, these findings were observed only at the highest exposure level. There was also a mild progressive decrease in pulmonary function, which was more consistent in females than males. Limited nasal epithelial changes resulted from NTDE exposure, including increases in minor olfactory epithelial degeneration, hyperplasia, and/or metaplasia. Increases in these findings were present primarily at the highest exposure level, and their minor and variable nature renders their biologic significance uncertain. Overall, the findings of this study demonstrated markedly less severe biologic responses to NTDE than observed previously in rats exposed similarly to TDE. Further, the effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors.

NOS-2 Inhibition in Phosgene-Induced Acute Lung Injury.

Phosgene exposure via an industrial or warfare release produces severe acute lung injury (ALI) with high mortality, characterized by massive pulmonary edema, disruption of epithelial tight junctions, surfactant dysfunction, and oxidative stress. There are no targeted treatments for phosgene-induced ALI. Previous studies demonstrated that nitric oxide synthase 2 (NOS-2) is upregulated in the lungs after phosgene exposure; however, the role of NOS-2 in the pathogenesis of phosgene-induced ALI remains unknown. We previously demonstrated that NOS-2 expression in lung epithelium exacerbates inhaled endotoxin-induced ALI in mice, mediated partially through downregulation of surfactant protein B (SP-B) expression. Therefore, we hypothesized that a selective NOS-2 inhibitor delivered to the lung epithelium by inhalation would mitigate phosgene-induced ALI. Inhaled phosgene produced increases in bronchoalveolar lavage fluid protein, histologic lung injury, and lung NOS-2 expression at 24 h. Administration of the selective NOS-2 inhibitor 1400 W via inhalation, but not via systemic delivery, significantly attenuated phosgene-induced ALI and preserved epithelial barrier integrity. Furthermore, aerosolized 1400 W augmented expression of SP-B and prevented downregulation of tight junction protein zonula occludens 1 (ZO-1), both critical for maintenance of normal lung physiology and barrier integrity. We also demonstrate for the first time that NOS-2-derived nitric oxide downregulates the ZO-1 expression at the transcriptional level in human lung epithelial cells, providing a novel target for ameliorating vascular leak in ALI. Our data demonstrate that lung NOS-2 plays a critical role in the development of phosgene-induced ALI and suggest that aerosolized NOS-2 inhibitors offer a novel therapeutic strategy for its treatment.

High sputum total adiponectin is associated with low odds for asthma.

OBJECTIVE: Adipose tissue produces adiponectin, an anti-inflammatory protein. High systemic total adiponectin is associated with a low risk for incident asthma but the association with lung adiponectin is not known. Our objective was to evaluate the association between sputum total adiponectin and asthma. METHODS: This case-control study included 44 cases with objectively-confirmed asthma and an equal number of body mass index (BMI) and sex-matched controls. Serum and sputum adiponectin were estimated by ELISA and Western Blot technique, respectively. While Fisher's exact test, t-test and Spearman's correlations were used for univariate analyses, Spearman and regression analyses were performed for multivariable analyses. RESULTS: While high-molecular-weight adiponectin was the dominant isoform in serum, medium-molecular-weight isoform was dominant in sputum. Sputum total adiponectin was not correlated with serum adiponectin or BMI. Sputum total adiponectin was lower among asthmatics than controls (p = 0.03), although individual sputum isoforms were not similarly associated. High sputum total adiponectin was associated with lower odds for asthma (OR 0.33, 95% C.I. 0.12, 0.91), even after adjustment for systemic adiposity measures including serum adiponectin. CONCLUSIONS: High sputum total adiponectin predicted lower odds for asthma, even after adjustment for serum adiponectin. Although not studied, it is possible that pharmacological modulation of sputum adiponectin may suggest new ways to prevent and/or treat asthma.

Effect of allergen inhalation on airway oxidant stress, using exhaled breath condensate 8-isoprostane, in mild asthma.

OBJECTIVE: Exhaled breath condensate (EBC) 8-isoprostane concentrations are increased in asthma, but it is not known if they acutely change following bronchoprovocation. The objective of this study was to evaluate EBC 8-isoprostane concentrations following allergen-induced bronchoprovocation in asthma. METHODS: This comparison study included eight mild atopic asthmatics and six controls. Asthmatics were challenged with inhaled specific allergen, methacholine, and irrelevant allergen in random order. Controls were challenged with irrelevant allergen. EBCs collected at 0, 3, 6, 9, and 23 hours by the R-tube method were measured for 8-isoprostanes by ELISA technique. Repeated measures ANOVA technique was used for analysis. RESULTS: EBC 8-isoprostane concentrations did not change following any inhalational challenge, as compared to baseline, in either asthmatics or controls. CONCLUSIONS: EBC 8-isoprostane concentrations do not acutely change following bronchoprovocation in subjects with mild asthma.

Part 1. Biologic responses in rats and mice to subchronic inhalation of diesel exhaust from U.S. 2007-compliant engines: report on 1-, 3-, and 12-month exposures in the ACES bioassay.

The Health Effects Institute and its partners conceived and funded a program to characterize the emissions from heavy-duty diesel engines compliant with the 2007 and 2010 on-road emissions standards in the United States and to evaluate indicators of lung toxicity in rats and mice exposed repeatedly to diesel exhaust (DE*) from 2007-compliant engines. The preliminary hypothesis of this Advanced Collaborative Emissions Study (ACES) was that 2007-compliant on-road diesel emissions ". . . will not cause an increase in tumor formation or substantial toxic effects in rats and mice at the highest concentration of exhaust that can be used . . . although some biological effects may occur." This hypothesis is being tested at the Lovelace Respiratory Research Institute (LRRI) by exposing rats by chronic inhalation as a carcinogenicity bioassay, measuring indicators of pulmonary toxicity in rats after 1, 3, 12, and 24-30 months of exposure (final time point depends on the survival of animals), and measuring similar indicators of pulmonary toxicity in mice after 1 and 3 months of exposure. This report provides results of exposures through 3 months in rats and mice. Emissions from a 2007-compliant, 500-horsepower-class engine and aftertreatment system operated on a variable-duty cycle were used to generate the animal inhalation test atmospheres. Four treatment groups were exposed to one of three concentrations (dilutions) of exhaust combined with crankcase emissions, or to clean air as a negative control. Dilutions of exhaust were set to yield average integrated concentrations of 4.2, 0.8, and 0.1 ppm nitrogen dioxide (NO2). Exposure atmospheres were analyzed by daily measurements of key components and periodic detailed physical-chemical characterizations. Exposures were conducted 16 hr/dy (overnight), 5 dy/wk. Rats were evaluated for hematology, serum chemistry, bronchoalveolar lavage (BAL), lung cell proliferation, and histopathology after 1 month of exposure, and the same indicators plus pulmonary function after 3 months. Mice were evaluated for BAL, lung cell proliferation, and respiratory tract histopathology after 1 month of exposure, and the same indicators plus hematology and serum chemistry after 3 months. Samples from both species were collected for ancillary studies performed by investigators who were not at LRRI and were funded separately. Exposures were accomplished as planned, with average integrated exposure concentrations within 20% of the target dilutions. The major components were the gaseous inorganic compounds, nitrogen monoxide (NO), NO2, and carbon monoxide (CO). Minor components included low concentrations of diesel particulate matter (DPM) and volatile and semivolatile organic compounds (VOCs and SVOCs). There were no exposure-related differences in mortality or clinically evident morbidity. Among the more than 100 biologic response variables evaluated, the majority showed no significant difference from control as a result of exposure to DE. There was evidence of early lung changes in the rats, accompanied by a number of statistically significant increases in inflammatory and oxidative stress indicators, and some evidence of subtle changes in pulmonary function. In general, statistically significant effects were observed only at the highest exposure level. The mice did not have the same responses as the rats, but did have small but statistically significant increases in lavage neutrophils and the cytokine IL-6 at 1 month (but not at 3 months). These findings suggest that the rats were more sensitive than mice to the subchronic exposures.

Effects of guaifenesin, N-acetylcysteine, and ambroxol on MUC5AC and mucociliary transport in primary differentiated human tracheal-bronchial cells.

BACKGROUND: Therapeutic intervention in the pathophysiology of airway mucus hypersecretion is clinically important. Several types of drugs are available with different possible modes of action. We examined the effects of guaifenesin (GGE), N-acetylcysteine (NAC) and ambroxol (Amb) on differentiated human airway epithelial cells stimulated with IL-13 to produce additional MUC5AC. METHODS: After IL-13 pre-treatment (3 days), the cultures were treated with GGE, NAC or Amb (10-300 µM) in the continued presence of IL-13. Cellular and secreted MUC5AC, mucociliary transport rates (MTR), mucus rheology at several time points, and the antioxidant capacity of the drugs were assessed. RESULTS: IL-13 increased MUC5AC content (~25%) and secretion (~2-fold) and decreased MTR, but only slightly affected the G' (elastic) or G" (viscous) moduli of the secretions. GGE significantly inhibited MUC5AC secretion and content in the IL-13-treated cells in a concentration-dependent manner (IC50s at 24 hr ~100 and 150 µM, respectively). NAC or Amb were less effective. All drugs increased MTR and decreased G' and G" relative to IL-13 alone. Cell viability was not affected and only NAC exhibited antioxidant capacity. CONCLUSIONS: Thus, GGE effectively reduces cellular content and secretion of MUC5AC, increases MTR, and alters mucus rheology, and may therefore be useful in treating airway mucus hypersecretion and mucostasis in airway diseases.

Effect of guaifenesin on mucin production, rheology, and mucociliary transport in differentiated human airway epithelial cells.

Guaifenesin is widely used to alleviate symptoms of excessive mucus accumulation in the respiratory tract. However, its mechanism of action is poorly understood. The authors hypothesized that guaifenesin improves mucociliary clearance in humans by reducing mucin release, by decreasing mucus viscoelasticity, and by increasing mucociliary transport. To test these hypotheses, human differentiated airway epithelial cells, cultured at an air-liquid interface, were treated with clinically relevant concentrations of guaifenesin by addition to the basolateral medium. To evaluate the effect on mucin secretion, the authors used an anzyme-linked immunosorbent assay (ELISA) to measure the amounts of MUC5AC protein in apical surface fluid and cell lysates. To measure mucociliary transportability, additional cultures were treated for 1 or 6 hours with guaifenesin, and the movement of cell debris was measured from video data. Further, the authors measured mucus dynamic viscoelasticity using a micro cone and plate rheometer with nondestructive creep transformation. Guaifenesin suppressed mucin production in a dose-dependent manner at clinically relevant concentrations. The reduced mucin production was associated with increased mucociliary transport and decreased viscoelasticity of the mucus. Viability of the cultures was not significantly affected. These results suggest that guaifenesin could improve mucociliary clearance in humans by reducing the release and/or production of mucins, thereby altering mucus rheology.

Fibrogenic and redox-related but not proinflammatory genes are upregulated in Lewis rat model of chronic silicosis.

Silicosis, a fibrotic granulomatous lung disease, may occur through accidental high-dose or occupational inhalation of silica, leading to acute/accelerated and chronic silicosis, respectively. While chronic silicosis has a long asymptomatic latency, lung inflammation and apoptosis are hallmarks of acute silicosis. In animal models, histiocytic granulomas develop within days after high-dose intratracheal (IT) silica instillation. However, following chronic inhalation of occupationally relevant doses of silica, discrete granulomas resembling human silicosis arise months after the final exposure without significant lung inflammation/apoptosis. To identify molecular events associated with chronic silicosis, lung RNA samples from controls or subchronic silica-exposed rats were analyzed by Affymetrix at 28 wk after silica exposures. Results suggested a significant upregulation of 144 genes and downregulation of 7 genes. The upregulated genes included complement cascade, chemokines/chemokine receptors, G-protein signaling components, metalloproteases, and genes associated with oxidative stress. To examine the kinetics of gene expression relevant to silicosis, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), Luminex-bead assays, Western blotting, and/or zymography were performed on lung tissues from 4 d, 28 wk, and intermediate times after subchronic silica exposure and compared with 14-d acute silicosis samples. Results indicated that genes regulating fibrosis (secreted phosphoprotein-1, Ccl2, and Ccl7), redox enzymes (superoxide dismutase-2 and arginase-1), and the enzymatic activities of matrix metalloproteinases 2 and 9 were upregulated in acute and chronic silicosis models. However, proinflammatory cytokines were strongly upregulated only in acute silicosis. Thus, inflammatory cytokines are associated with acute but not chronic silicosis. Data suggest that genes regulating fibrosis, oxidative stress, and metalloproteases may contribute to both acute and chronic silicosis.

Antioxidant diet protects against emphysema, but increases mortality in cigarette smoke-exposed mice.

Oxidative stress plays an important role in cigarette smoke-induced lung inflammation and emphysema. We produced an enriched diet by adding freeze-dried fruits and vegetables and additional supplements to the 8604 Teklad Rodent Diet, a standard rodent diet. In this study, we examined the effects of the antioxidant-enriched diet on cigarette smoke-induced lung inflammation and emphysema. CH3/HeN mice were fed either a regular diet or the supplemented diet. These mice were exposed to filtered air, a low concentration of cigarette smoke (total particulate matter: 100 mg/m3) or a high concentration of cigarette smoke (total particulate matter: 250 mg/m3) for 6 h/day, 5 days/week for total 16 weeks. Surprisingly, increased mortality (53%) was observed in the high concentration of cigarette smoke-exposed mice fed the antioxidant diet compared to the high concentration of cigarette smoke-exposed mice that were fed a regular diet (13%). The necropsy analysis revealed nasal passage obstruction due to mucous plugging in cigarette smoke-exposed mice on the antioxidant diet. However, the antioxidant diet significantly reduced neutrophilic inflammation and emphysema in the high concentration of cigarette smoke-exposed mice as compared to the regular diet /high concentration of cigarette smoke controls. The antioxidant capacity in the bronchoalveolar fluid or oxidative damage to the lung tissue was not affected by the antioxidant diet. Pro-MMP-2, MMP-2, and MMP-9 activity did not correlate with the protective effects of AOD on cigarette smoke-induced emphysema. These data suggest that the antioxidant diet reduced cigarette smoke-induced inflammation and emphysema, but increased mortality in the obligate nose-breathing mice.

Health effects of subchronic inhalation exposure to simulated downwind coal combustion emissions.

CONTEXT: There have been no animal studies of the health effects of repeated inhalation of mixtures representing downwind pollution from coal combustion. Environmental exposures typically follow atmospheric processing and mixing with pollutants from other sources. OBJECTIVE: This was the fourth study by the National Environmental Respiratory Center to create a database for responses of animal models to combustion-derived pollutant mixtures, to identify causal pollutants-regardless of source. METHODS: F344 and SHR rats and A/J, C57BL/6, and BALB/c mice were exposed 6 h/day 7 days/week for 1 week to 6 months to three concentrations of a mixture simulating key components of "downwind" coal combustion emissions, to the highest concentration filtered to remove particulate matter (PM), or to clean air. Emissions from low-sulfur subbituminous coal were modified to create a mixture recommended by an expert workshop. Sulfur dioxide, nitrogen oxides, and PM were the dominant components. Nonanimal-derived PM mass concentrations of nominally 0, 100, 300, and 1000 µg/m(3) were mostly partially neutralized sulfate. RESULTS: Only 17 of 270 species-gender-time-outcome comparisons were significantly affected by exposure; some models showed no effects. There was strong evidence that PM participated meaningfully in only three responses. CONCLUSION: On a total mass or PM mass basis, this mixture was less toxic overall than diesel and gasoline exhausts or wood smoke. The largely sulfate PM contributed to few effects and was the sole cause of none. The study did not allow identification of causal pollutants, but the potential role of NOx in some effects is suggested by the literature.

Engine-operating load influences diesel exhaust composition and cardiopulmonary and immune responses.

BACKGROUND: The composition of diesel engine exhaust (DEE) varies by engine type and condition, fuel, engine operation, and exhaust after treatment such as particle traps. DEE has been shown to increase inflammation, susceptibility to infection, and cardiovascular responses in experimentally exposed rodents and humans. Engines used in these studies have been operated at idle, at different steady-state loads, or on variable-load cycles, but exposures are often reported only as the mass concentration of particulate matter (PM), and the effects of different engine loads and the resulting differences in DEE composition are unknown. OBJECTIVES: We assessed the impacts of load-related differences in DEE composition on models of inflammation, susceptibility to infection, and cardiovascular toxicity. METHODS: We assessed inflammation and susceptibility to viral infection in C57BL/6 mice and cardiovascular toxicity in APOE-/- mice after being exposed to DEE generated from a single-cylinder diesel generator operated at partial or full load. RESULTS: At the same PM mass concentration, partial load resulted in higher proportions of particle organic carbon content and a smaller particle size than did high load. Vapor-phase hydrocarbon content was greater at partial load. Compared with high-load DEE, partial-load DEE caused greater responses in heart rate and T-wave morphology, in terms of both magnitude and rapidity of onset of effects, consistent with previous findings that systemic effects may be driven largely by the gas phase of the exposure atmospheres. However, high-load DEE caused more lung inflammation and greater susceptibility to viral infection than did partial load. CONCLUSIONS: Differences in engine load, as well as other operating variables, are important determinants of the type and magnitude of responses to inhaled DEE. PM mass concentration alone is not a sufficient basis for comparing or combining results from studies using DEE generated under different conditions.

Time course of lesion development in the hairless guinea-pig model of sulfur mustard-induced dermal injury.

The objective of these studies was to provide detailed analyses of the time course of sulfur mustard (SM) vapor-induced clinical, histological, and biochemical changes following cutaneous exposure in hairless guinea-pigs. Three 6 cm(2) sites on the backs of each guinea-pig were exposed to SM vapor (314 mg(3) ) for 6 minutes (low dose) or 12 minutes (high dose). Animals were killed at 6, 24, and 48 hours, or 2 weeks postexposure. Erythema, edema, histopathology, and analysis of matrix metalloproteinase (MMP)-2 and -9 content were evaluated. Erythema was observed by 6 hours, and edema by 24 hours postexposure. Vapor exposure caused epidermal necrosis with varying degrees of dermatitis, ulceration, hemorrhage, and separation of the dermis from the epidermis. Later changes included epidermal regeneration with hyperplasia and formation of granulation tissue in the dermis with loss of hair follicles and glandular structures. Relative amounts of pro and active MMP-2 and MMP-9 were significantly increased in the high-dose SM group at 2 weeks. Erythema, edema, and histologic changes are consistent with findings among human victims of SM attack. This model, with observations to 2 weeks, will be useful in assessing the efficacy of countermeasures against SM.

Sulfur mustard vapor effects on differentiated human lung cells.

CONTEXT: Sulfur mustard (SM) causes skin blistering and long-term pulmonary dysfunction. Its adverse effects have been studied in battlefield-exposed humans, but lack of knowledge regarding confounding factors makes interpretation challenging. Animal studies are critical to understanding mechanisms, but differences between animals and humans must be addressed. Studies of cultured human cells can bridge animal studies and humans. OBJECTIVE: Evaluate effects of SM vapor on airway cells. MATERIALS AND METHODS: We examined responses of differentiated human tracheal/bronchial epithelial cells, cultured at an air-liquid interface, to SM vapors. SM effects on metabolic activity (Water Soluble Tetrazolium (WST) assay), cytokine and metalloproteinase secretion, and cellular heme oxygenase 1 (HO-1), an oxidative stress indicator, were measured after 24 h. RESULTS: At noncytotoxic levels of exposure, interleukin 8 and matrix metalloproteinase-13 were significantly increased in these cultures, but HO-1 was not significantly affected. DISCUSSION AND CONCLUSION: Exposure of differentiated airway epithelial cells to sub-cytotoxic levels of SM vapor induced inflammatory and degradative responses that could contribute to the adverse health effects of inhaled SM.

Atmospheric transformation of diesel emissions.

The hypothesis of this study was that exposing diesel exhaust (DE*) to the atmosphere transforms its composition and toxicity. Our specific aims were (1) to characterize the gas- and particle-phase products of atmospheric transformations of DE under the influence of daylight, ozone (O3), hydroxyl (OH) radicals, and nitrate (NO3) radicals; and (2) to explore the biologic activity of DE before and after the transformations took place. The study was executed with the aid of the EUPHORE (European Photoreactor) outdoor simulation chamber facility in Valencia, Spain. EUPHORE is one of the largest and best-equipped facilities of its kind in the world, allowing investigation of atmospheric transformation processes under realistic ambient conditions (with dilutions in the range of 1:300). DE was generated on-site using a modern light-duty diesel engine and a dynamometer system equipped with a continuous emission-gas analyzer. The engine (a turbocharged, intercooled model with common-rail direct injection) was obtained from the Ford Motor Company. A first series of experiments was carried out in January 2005 (the winter 2005 campaign), a second in May 2005 (the summer 2005 campaign), and a third in May and June 2006 (the summer 2006 campaign). The diesel fuel that was used closely matched the one currently in use in most of the United States (containing 47 ppm sulfur and 15% aromatic compounds). Our experiments examined the effects on the composition of DE aged in the dark with added NO3 radicals and of DE aged in daylight with added OH radicals both with and without added volatile organic compounds (VOCs). In order to remove excess nitrogen oxides (NO(x)), a NO(x) denuder was devised and used to conduct experiments in realistic low-NO(x) conditions in both summer campaigns. A scanning mobility particle sizer was used to determine the particle size and the number and volume concentrations of particulate matter (PM) in the DE. O3, NO(x), and reactive nitrogen oxides (NO(y)) were monitored using chemiluminescence and Fourier transform infrared instruments. At the end of the exposures, samples of particle-associated and semivolatile organic compounds (SVOCs) were collected from the chamber for chemical analysis using an XAD-coated annular denuder followed by a filter and XAD cartridge. (XAD is an adsorbent polystyrene divinylbenzene resin used in sampling cartridges.) Samples for toxicity testing were collected using Teflon filters followed by two XAD cartridges. The chemical analyses included determination of organic carbon (OC), elemental carbon (EC), carbon fractions, inorganic ions (e.g., sulfate and nitrate), and speciated organic compounds (polycyclic aromatic hydrocarbons [PAHs], nitro-PAHs, polar compounds, alkanes, hopanes, and steranes). The toxicity tests were performed with extracts of PM combined with the SVOCs. The biologic activity of these extracts was evaluated in vivo by instilling them into the tracheas of rodents and measuring pulmonary toxicity, inflammation, and oxidative-stress responses. Results from the chemical analyses indicated that aging DE in the dark with added NO3 radicals and aging DE in daylight with and without additions led to the formation of additional particles and SVOC mass caused by reactions of VOCs, SVOCs, and inorganic gases. The greatest increase in mass occurred with the addition of VOCs as co-reactants. The proportions of the pyrolized OC (POC) fraction increased in the organic mass, which suggested that highly polar and oligomeric compounds had been formed. Results from the toxicity testing were consistent with the hypothesis that the toxicity of the samples had been affected by changes in their composition (caused both by the atmospheric aging and by changes in the initial composition of the DE presumably associated with changes in the engine, which was new at the outset and accrued wear during the study).

Cardiopulmonary response to inhalation of biogenic secondary organic aerosol.

An irradiation chamber designed for reproducible generation of inhalation test atmospheres of secondary organic aerosol (SOA) was used to evaluate cardiopulmonary responses in rodents exposed to SOA derived from the oxidation of alpha-pinene. SOA atmospheres were produced with 10:1 ratios of alpha-pinene:nitrogen oxides (NO(x)) and 10:1:1 ratios of alpha-pinene:nitrogen oxides:sulfur dioxide (SO(2)). SOA atmospheres were produced to yield 200 microg m(-3) of particulate matter (PM). Exposures were conducted downstream of honeycomb denuders employed to remove the gas-phase precursors and reaction products. Nose-only exposures were conducted with both rats (pulmonary effects) and mice (pulmonary and cardiovascular effects). Composition of the atmospheres was optimized to ensure that the SOA generated resembled SOA observed in previous irradiation studies, and contained specific SOA compounds of interest (e.g., organosulfates) identified in ambient air. Pulmonary and cardiovascular toxicity were measured in two different rodent species. In situ chemiluminescence and thiobarbituric acid- reactive substances (TBARS) were used to evaluate oxidative reactions in the F344 rats. ApoE(-/-) mice were exposed for 7 days and measurements of TBARS and gene expression of heme oxygenase-1 (HO-1), endothelin-1 (ET-1), matrix metalloproteinase-9 (MMP-9) were made in aorta. Pulmonary inflammatory responses in both species were measured by bronchoalveolar lavage fluid (BALF) cell counts. No pulmonary inflammation was observed in either species. A mild response was observed in mouse aorta for the upregulation of HO-1 and MMP-9, but was not seen for ET-1. Overall, alpha-pinene-derived SOA, including SOA that included organosulfate compounds, revealed limited biological response after short-term inhalation exposures.

Effects of 10 cigarette smoke condensates on primary human airway epithelial cells by comparative gene and cytokine expression studies.

Cigarettes vary in tobacco blend, filter ventilation, additives, and other physical and chemical properties, but little is known regarding potential differences in toxicity to a smoker's airway epithelia. We compared changes in gene expression and cytokine production in primary normal human bronchial epithelial cells following treatment for 18 h with cigarette smoke condensates (CSCs) prepared from five commercial and four research cigarettes, at doses of approximately 4 microg/ml nicotine. Nine of the CSCs were produced under a standard International Organization for Standardization smoking machine regimen and one was produced by a more intense smoking machine regimen. Isolated messenger RNA (mRNA) was analyzed by microarray hybridization, and media was analyzed for secreted cytokines and chemokines. Twenty-one genes were differentially expressed by at least 9 of the 10 CSCs by more than twofold, including genes encoding detoxifying and antioxidant proteins. Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and NAD(P)H dehydrogenase, quinone 1 (NQO-1) were selected for validation with quantitative real-time PCR (qRT-PCR) and Western blot analyses. NQO-1 expression determined with microarrays, qRT-PCR, and Western blotting differed among the CSC types, with good correlation among the different assays. CYP1A1 mRNA levels varied substantially, but there was little correlation with the protein levels. For each CSC, the three most induced and three most repressed genes were identified. These genes may be useful as markers of exposure to that particular cigarette type. Furthermore, differences in interleukin-8 secretion were observed. These studies lay the foundation for future investigations to analyze differences in the responses of in vivo systems to tobacco products marketed with claims of reduced exposure or reduced harm.

Sulfur mustard induces immune sensitization in hairless guinea pigs.

Sulfur mustard (SM, bis-(2-chloroethyl) sulfide) is a well known chemical warfare agent that may cause long-term debilitating injury. Because of the ease of production and storage, it has a strong potential for chemical terrorism; however, the mechanism by which SM causes chronic tissue damage is essentially unknown. SM is a potent protein alkylating agent, and we tested the possibility that SM modifies cellular antigens, leading to an immunological response to "altered self" and a potential long-term injury. To that end, in this communication, we show that dermal exposure of euthymic hairless guinea pigs induced infiltration of both CD4(+) and CD8(+) T cells into the SM-exposed skin and strong upregulated expression of proinflammatory cytokines and chemokines (TNF-alpha, IFN-gamma, and IL-8) in distal tissues such as the lung and the lymph nodes. Moreover, we present evidence for the first time that SM induces a specific delayed-type hypersensitivity response that is associated with splenomegaly, lymphadenopathy, and proliferation of cells in these tissues. These results clearly suggest that dermal exposure to SM leads to immune activation, infiltration of T cells into the SM-exposed skin, delayed-type hypersensitivity response, and molecular imprints of inflammation in tissues distal from the site of SM exposure. These immunological responses may contribute to the long-term sequelae of SM toxicity.