A novel large animal model of smoke inhalation-induced acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is multifactorial and can result from sepsis, trauma, or pneumonia, amongst other primary pathologies. It is one of the major causes of death in critically ill patients with a reported mortality rate up to 45%. The present study focuses on the development of a large animal model of smoke inhalation-induced ARDS in an effort to provide the scientific community with a reliable, reproducible large animal model of isolated toxic inhalation injury-induced ARDS. METHODS: Animals (n = 21) were exposed to smoke under general anesthesia for 1 to 2 h (median smoke exposure = 0.5 to 1 L of oak wood smoke) after the ultrasound-guided placement of carotid, pulmonary, and femoral artery catheters. Peripheral oxygen saturation (SpO2), vital signs, and ventilator parameters were monitored throughout the procedure. Chest x-ray, carotid, femoral and pulmonary artery blood samples were collected before, during, and after smoke exposure. Animals were euthanized and lung tissue collected for analysis 48 h after smoke inhalation. RESULTS: Animals developed ARDS 48 h after smoke inhalation as reflected by a decrease in SpO2 by approximately 31%, PaO2/FiO2 ratio by approximately 208 (50%), and development of bilateral, diffuse infiltrates on chest x-ray. Study animals also demonstrated a significant increase in IL-6 level, lung tissue injury score and wet/dry ratio, as well as changes in other arterial blood gas (ABG) parameters. CONCLUSIONS: This study reports, for the first time, a novel large animal model of isolated smoke inhalation-induced ARDS without confounding variables such as cutaneous burn injury. Use of this unique model may be of benefit in studying the pathophysiology of inhalation injury or for development of novel therapeutics.

Origami paper analytical assay based on metal complex sensor for rapid determination of blood cyanide concentration in fire survivors.

Cyanide-based blood poisoning can seriously damage fire victims and cause death if not detected quickly. Previous conventional methods require laboratory equipment, which are expensive and increase the duration of the analysis. Here, a simple origami based microfluidic device was introduced for point of need detection of blood cyanide concentration in people involved in fire. The device is made of four layers of paper. Each layer was in the size of 1 × 1 cm folded on each other. In this work, the blood sample was acidified by trichloroacetic acid to separate cyanide from methaemoglobin in the form of HCN gas. The produced gas released into borate buffer to recover free cyanide ions which interacted with the Pt complex ([Pt(p-MeC6H4)2(phen)]) used as a receptor in this study. Optimized conditions were applied to have a suitable interaction causing the color of the receptor to change from yellow to colorless. The color changes were recorded by a smartphone, and the sensor response was calculated by the routine image analysis software. The assay was capable of determining cyanide ions at different concentrations in the range of 1.0 to 100.0 µmol L-1. The detection limit of these determination was equal to 0.4 µmol L-1. The assay responses were not affected by the interfering species. As a practical analysis, the proposed sensor was applied to determine cyanide ions in the blood sample of 20 studied fire survivors and 10 controls with high accuracy.

Effects of simvastatin on iNOS and caspase­3 levels and oxidative stress following smoke inhalation injury.

The overexpression of inducible nitric oxide synthase (iNOS) induces cell apoptosis through various signal transduction pathways and aggravates lung injury. Caspase­3 is an important protein in the apoptotic pathway and its activation can exacerbate apoptosis. Simvastatin, a hydroxymethyl glutaryl­A reductase inhibitor, protects against smoke inhalation injury by inhibiting the synthesis and release of inflammatory factors and decreasing cell apoptosis. Following the establishment of an animal model of smoke inhalation injury, lung tissue and serum were collected at different time points and the protein and mRNA expression of iNOS and caspase­3 in lung tissue by immunochemistry, western blot and reverse transcription­quantitative polymerase chain reaction, the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in lung tissue and serum were analyzed using thiobarbituric acid method and the WST­1 method. The results were statistically analyzed. The lung tissues of the rats in the saline group and the low­, middle­ and high­dose groups exhibited clear edema and hemorrhage, and had significantly higher pathological scores at the various time points compared with the rats in the control group (P<0.05). Furthermore, lung tissue and serum samples obtained from these four groups had significantly higher mRNA and protein expression levels of iNOS and caspase­3 (P<0.05), significantly lower SOD activity and higher MDA content (P<0.05). Compared with the saline group, the low­, middle­ and high­dose groups had significantly lower pathological scores (P<0.05), significantly lower mRNA and protein expression levels of iNOS, caspase­3 and MDA content in lung tissues (P<0.05) and significantly higher SOD activity in lung tissues and serum. The middle­ and high­dose groups had significantly lower pathological scores (P<0.05), significantly decreased iNOS and caspase­3 mRNA and protein expression in lung tissues, significantly higher SOD activity in lung tissues and serum and a significantly lower MDA content (P<0.05) compared with the low­dose group. With the exception of SOD activity in lung tissues at 24 and 72 h and MDA content in serum at 48 h, no significant differences were observed between the middle­ and high­dose groups. The present study demonstrated that there was an association between the therapeutic effect and dosage of simvastatin within a definitive range. In rats with smoke inhalation injury, simvastatin inhibited iNOS and caspase­3 expression in lung tissues and mitigated oxidative stress, thereby exerting a protective effect. In addition, the effect and dose were associated within a definitive range.

MicroRNA Let-7f-1-3p attenuates smoke-induced apoptosis in bronchial and alveolar epithelial cells in vitro by targeting FOXO1.

Bronchial and alveolar epithelial cell apoptosis is a vital step in smoke-induced lung injury. We investigated whether and how microRNA (miRNA) Let-7f-1-3p would regulate smoke-induced apoptosis in bronchial and alveolar epithelial cells. Human small airway epithelial cells (HSAEC) and human pulmonary alveolar epithelial cells (HPAEpiC) were cultured using an air-liquid interface cell culture system. These cells were treated with Let-7f-1-3p agomir or antagomir for 24 h before smoke exposure or sham operation, after which the cells were rinsed and cultured for 24 h before cell viability, apoptosis, cytolysis, Caspase-9/8/3 activity assays, quantitative real-time polymerase chain reaction and Western blot. Bioinformatic and luciferase reporter assays were performed to predict or verify the target gene of Let-7f-1-3p. We found that smoke exposure significantly reduced Let-7f-1-3p expression level in HSAEC and HPAEpiC. Let-7f-1-3p agomir significantly attenuated cell apoptosis, cytolysis and Caspase-3, -8 and -9 activation while rescuing cell viability of smoke-exposed HSAEC and HPAEpiC. Let-7f-1-3p agomir downregulated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL) and B-cell lymphoma-2 (Bcl2)-like protein 11 (Bim) protein level in HSAEC and HPAEpiC. Forkhead box-O1 (FOXO1) was verified as a putative regulatory target of Let-7f-1-3p. Smoke exposure increased FOXO1 mRNA and protein level in HSAEC and HPAEpiC, which was attenuated by Let-7f-1-3p agomir treatment. FOXO1 inhibition by small-molecule drug partially attenuated the increase in smoke-exposed HSAEC and HPAEpiC apoptosis, cytolysis and the decrease in cell viability caused by Let-7f-1-3p antagomir treatment. We concluded Let-7f-1-3p attenuated smoke-induced apoptosis in HSAEC and HPAEpiC by targeting FOXO1.

Identification of differentially expressed proteins in the injured lung from zinc chloride smoke inhalation based on proteomics analysis.

BACKGROUND: Lung injury due to zinc chloride smoke inhalation is very common in military personnel and leads to a high incidence of pulmonary complications and mortality. The aim of this study was to uncover the underlying mechanisms of lung injury due to zinc chloride smoke inhalation using a rat model. METHODS: Histopathology analysis of rat lungs after zinc chloride smoke inhalation was performed by using haematoxylin and eosin (H&E) and Mallory staining. A lung injury rat model of zinc chloride smoke inhalation (smoke inhalation for 1, 2, 7 and 14 days) was developed. First, isobaric tags for relative and absolute quantization (iTRAQ) and weighted gene co-expression network analysis (WGCNA) were used to identify important differentially expressed proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to study the biological functions of differentially expressed proteins. Then, analysis of lung injury repair-related differentially expressed proteins in the early (day 1 and day 2) and middle-late stages (day 7 and day 14) of lung injury after smoke inhalation was performed, followed by the protein-protein interaction (PPI) analysis of these differentially expressed proteins. Finally, the injury repair-related proteins PARK7 and FABP5 were validated by immunohistochemistry and western blot analysis. RESULTS: Morphological changes were observed in the lung tissues after zinc chloride smoke inhalation. A total of 27 common differentially expressed proteins were obtained on days 1, 2, 7 and 14 after smoke inhalation. WGCNA showed that the turquoise module (which involved 909 proteins) was most associated with smoke inhalation time. Myl3, Ckm, Adrm1 and Igfbp7 were identified in the early stages of lung injury repair. Gapdh, Acly, Tnni2, Acta1, Actn3, Pygm, Eno3 and Tpi1 (hub proteins in the PPI network) were identified in the middle-late stages of lung injury repair. Eno3 and Tpi1 were both involved in the glycolysis/gluconeogenesis signalling pathway. The expression of PARK7 and FABP5 was validated and was consistent with the proteomics analysis. CONCLUSION: The identified hub proteins and their related signalling pathways may play crucial roles in lung injury repair due to zinc chloride smoke inhalation.

A Comparative Histological Study of Submucosal Gland Hypertrophy in Trachea of Mice Exposed to Cigarette and Shisha Smoke.

OBJECTIVE: To observe the effects of Shisha smoke on submucosal glands of trachea of mice; and compare it with tracheal glands of mice exposed to cigarette smoke. STUDY DESIGN: Randomised controlled trial. PLACE AND DURATION OF STUDY: Department of Anatomy, Islamic International Medical College, Rawalpindi, in collaboration with National Institute of Health (NIH), Islamabad from October 2013 till April 2014. METHODOLOGY: Sample comprised of 40 adult male mice of strain BALB/c. They were randomly divided into three groups. Control group was labelled as Group 'C'. The mice in this group were kept in a whole body smoke exposure chamber and were exposed to fresh air. Shisha group was labelled as Group 'SS', and the mice in this group were exposed to Shisha smoke. Mice in the third group labelled as Group CS were exposed to cigarette smoke. All the mice were dissected after an exposure period of eight weeks. Tracheal tissue was stained and examined microscopically for submucosal gland hypertrophy and compared with the control group, using Reid's Index. An Index of more than 0.4 is termed as hypertrophy. RESULTS: There was significant submucosal gland hypertrophy in groups CS and SS as compared to group C. There was also significant difference in the frequency of mucosal hypertrophy between SS (93.7%) and CS groups (53.3%), which was found statistically significant (p<0.001). CONCLUSION: Shisha smoking was significantly associated with mucosal hypertrophy when compared with cigarette smoking and controls. Shishasmoke contains higher level of toxicants as compared to cigarette smoke, and it causes more oxidative damage of tissues.

A Curious Case of Inhalation Fever Caused by Synthetic Cannabinoid.

BACKGROUND: This case report describes inhalation fever as an uncommon pulmonary adverse effect of synthetic cannabinoids. CASE REPORT: A 29-year-old man was brought in for severe agitation after smoking K2, a synthetic cannabinoid. He required multiple doses of lorazepam and haloperidol for sedation. His vital signs were notable for a mild fever and tachycardia. Otherwise, the rest of his exam was unremarkable. The laboratory test was significant for leucocytosis and diffuse reticular-nodular and interstitial infiltrates on chest radiograph. Urine drug toxicology was negative. Interestingly, his symptoms and pulmonary infiltrates on the chest radiograph resolved spontaneously after 24 hours of observation. CONCLUSIONS: This patient developed transient pulmonary infiltrates and fever following the synthetic cannabinoid inhalation, as seen in self-limiting inhalation fever. Inhalation fever as a consequence of synthetic cannabinoid has not been described previously and there is a need for further research in this field.

Low-dose theophylline restores corticosteroid responsiveness in rats with smoke-induced airway inflammation.

Patients with chronic obstructive pulmonary disease (COPD) respond poorly to corticosteroids. Histone deacetylase-2 (HDAC-2) plays a pivotal role in many cases of steroid insensitivity. The main aim of this study was to restore the smoking-induced reduction in corticosteroid sensitivity by increasing HDAC-2 activity using low-dose theophylline. Rats were exposed to cigarette smoke (CS) and treated with budesonide and two doses of theophylline. Besides the pathologic examination and cell counting in the bronchoalveolar lavage fluid (BALF), the expression of HDAC-2 and CXC chemokine ligand-8 (CXCL-8) were measured. Airway inflammation induced by CS was demonstrated by pathologic changes of lung tissue and increased level of CXCL-8. CS exposure also markedly decreased HDAC-2 expression. Moreover, a negative correlation was found between HDAC-2 activity and a lung destruction index. The index was restored to control levels with inhaled corticosteroid treatment in combination with a low, not a high, dose of theophylline. These results indicate that low-dose theophylline might provide protection from smoke damage and improve the anti-inflammatory effects of steroids by increasing HDAC-2 activity.

Malondialdehyde and superoxide dismutase correlate with FEV(1) in patients with COPD associated with wood smoke exposure and tobacco smoking.

Tobacco smoking is the primary risk factor for chronic obstructive pulmonary disease (COPD). However, recent epidemiological studies have established domestic exposure to wood smoke and other biomass fuels as additional important risk factors, characteristic in developing countries. Oxidative stress is one of the mechanisms concerned with pathogenesis of COPD. However, the molecular mechanisms involved in the onset and progress of COPD associated with biomass and specifically that derived from wood smoke exposure remain unknown. We analyzed the relationship between forced expiratory volume in first second (FEV(1)) with plasma malondialdehyde (MDA) concentration and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) in COPD patients associated with wood smoke (WSG; n = 30), tobacco smoking (TSG; n = 30), and healthy control subjects (HCG; n = 30). Differences between FEV(1) from WSG and TSG (58 +/- 22% and 51 +/- 24%, respectively) with HCG (100 +/- 6%) were observed (P < 0.01). Plasma MDA concentration was higher in both WSG and TSG (1.87 +/- 0.81 and 1.68 +/- 0.82 nmol/mL, respectively) compared with HCG (0.42 +/- 0.17 nmol/mL; P < 0.01). SOD activity showed a significant increase in both WSG and TSG (0.36 +/- 0.12 and 0.37 +/- 0.13 U/mL) compared with HCG (0.19 +/- 0.04 U/mL; P < 0.01). No differences were shown regarding GPx, GR, and GST activities between COPD and control groups. Inverse correlations were founded between MDA and SOD with FEV(1) in both COPD patients and control subjects (P < 0.001). These results indicate a role for oxidative stress in COPD associated with wood smoke similar to that observed with tobacco smoking in subjects who ceased at least 10 years previous to this study.

Cyanide poisoning and cardiac disorders: 161 cases.

BACKGROUND: Inhalation of hydrogen cyanide from smoke in structural fires is common, but cardiovascular function in these patients is poorly documented. OBJECTIVE: The objective was to study the cardiac complications of cyanide poisoning in patients who received early administration of a cyanide antidote, hydroxocobalamin (Cyanokit; Merck KGaA, Darmstadt, Germany [in the United States, marketed by Meridian Medical Technologies, Bristol, TN]). METHODS: The medical records of 161 fire survivors with suspected or confirmed cyanide poisoning were reviewed in an open, multicenter, retrospective review of cases from the Emergency Medical Assistance Unit (Service d'Aide Médical d'Urgence) in France. RESULTS: Cardiac arrest (61/161, 58 asystole, 3 ventricular fibrillation), cardiac rhythm disorders (57/161, 56 supraventricular tachycardia), repolarization disorders (12/161), and intracardiac conduction disorders (5/161) were observed. Of the total 161 patients studied, 26 displayed no cardiac disorder. All patients were given an initial dose of 5 g of hydroxocobalamin. Non-responders received a second dose of 5 g of hydroxocobalamin. Of the patients initially in cardiac arrest, 30 died at the scene, 24 died in hospital, and 5 survived without cardiovascular sequelae. Cardiac disorders improved with increasing doses of hydroxocobalamin, and higher doses of the antidote seem to be associated with a superior outcome in patients with initial cardiac arrest. CONCLUSIONS: Cardiac complications are common in cyanide poisoning in fire survivors.

Acute respiratory distress syndrome after zinc chloride inhalation: survival after extracorporeal life support and corticosteroid treatment.

No standard protocol exists for the treatment of acute respiratory distress syndrome induced by inhalation of smoke from a smoke bomb. In this case, a 23-year-old man was exposed to smoke from a smoke grenade for approximately 10 to 15 minutes without protective breathing apparatus. Acute respiratory distress syndrome developed subsequently, complicated by bilateral pneumothorax and pneumomediastinum 48 hours after inhalation. Despite mechanical ventilation and bilateral tube thoracostomy, the patient was severely hypoxemic 4 days after hospitalization. His condition improved upon treatment with high-dose corticosteroids, an additional 500-mg dose of methylprednisolone, and the initiation of extracorporeal life support. Arterial oxygenation decreased gradually after abrupt tapering of the corticosteroid dose and discontinuation of the life support. On day 16 of hospitalization, the patient experienced progressive deterioration of arterial oxygenation despite the intensive treatment. The initial treatment regimen (ie, corticosteroids and extracorporeal life support) was resumed, and the patient's arterial oxygenation improved. The patient survived.

California wildfires of 2008: coarse and fine particulate matter toxicity.

BACKGROUND: During the last week of June 2008, central and northern California experienced thousands of forest and brush fires, giving rise to a week of severe fire-related particulate air pollution throughout the region. California experienced PM(10-2.5) (particulate matter with mass median aerodynamic diameter > 2.5 mum to < 10 mum; coarse ) and PM(2.5) (particulate matter with mass median aerodynamic diameter < 2.5 mum; fine) concentrations greatly in excess of the air quality standards and among the highest values reported at these stations since data have been collected. OBJECTIVES: These observations prompt a number of questions about the health impact of exposure to elevated levels of PM(10-2.5) and PM(2.5) and about the specific toxicity of PM arising from wildfires in this region. METHODS: Toxicity of PM(10-2.5) and PM(2.5) obtained during the time of peak concentrations of smoke in the air was determined with a mouse bioassay and compared with PM samples collected under normal conditions from the region during the month of June 2007. RESULTS: Concentrations of PM were not only higher during the wildfire episodes, but the PM was much more toxic to the lung on an equal weight basis than was PM collected from normal ambient air in the region. Toxicity was manifested as increased neutrophils and protein in lung lavage and by histologic indicators of increased cell influx and edema in the lung. CONCLUSIONS: We conclude that the wildfire PM contains chemical components toxic to the lung, especially to alveolar macrophages, and they are more toxic to the lung than equal doses of PM collected from ambient air from the same region during a comparable season.

Biomass smoke induced bronchial anthracofibrosis: presenting features and clinical course.

BACKGROUND: The presenting features and clinical course of biomass smoke induced bronchial anthracofibrosis (BAF) are not well known. PATIENTS AND METHODS: 333 patients who had a history of long-term exposure to biomass smoke, having BAF confirmed by a bronchoscopy from January 1998 to December 2004, were included in this study. The clinical features, associated diseases, and clinical outcomes were investigated through the analysis of medical records. RESULTS: There were 51 males (15.3%) and 282 females (84.7%), having a mean age of 72.3 years, ranging from 47 to 90. 33% of patients had a past history of pulmonary tuberculosis. Dyspnea (38.4%) and cough (29.8%) were most common presenting symptoms, followed by hemoptysis (8.9%). Baseline pulmonary function showed mild airflow obstruction. Among patients with forced expiratory volume in 1s (FEV(1))/forced vital capacity (FVC)<0.7, Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I and II were most common. Associated diseases were active tuberculosis in 33.9% of patients, pneumonia in 29.5%, acute exacerbation of chronic airways disease in 22.5%, and malignancy in 4.8%. Among the 18 patients who died at a hospital during the follow-up period, acute infection and malignancy were common causes of death. CONCLUSIONS: These findings suggest that biomass smoke induced BAF usually appears clinically as a form of obstructive airways disease. Since various pulmonary diseases, including tuberculosis, pneumonia, and malignancy, can be associated with BAF, thorough clinical evaluation and close follow-up of these patients are required.

Wood smoke-associated lung disease: a clinical, functional, radiological and pathological description.

BACKGROUND: Approximately half of the world's population, and up to 90% of households in rural areas of developing countries, depend on biomass for cooking and heating. SETTING: The National Institute of Respiratory Diseases, México City. OBJECTIVE: To describe wood smoke-associated lung disease (WSLD). DESIGN: Description of the clinical, functional and radiological manifestations of patients with WSLD, and a comparison of pathological findings of patients who died of WSLD and smokers who died of chronic bronchitis. RESULTS: All patients with WSLD were non-smoking women with chronic bronchitis, in whom asthma, bronchiectasis, tuberculosis, congestive heart failure, extreme obesity and alfa-1 antitrypsin deficiency had been excluded. All patients used wood for cooking and had been exposed to wood smoke for a median of 45 years. Dyspnoea, airway obstruction, air trapping, increased airway resistance, pathological evidence of anthracosis, chronic bronchitis, centrilobular emphysema and pulmonary hypertension were present in most patients with WSLD. Bronchial squamous metaplasia was a common finding. There were no significant differences in the histopathological findings between patients with WSLD and smokers. Diffuse interstitial fibrosis was absent in all patients. CONCLUSIONS: Patients with WSLD have obstructive lung disease, chronic bronchitis, emphysema and pulmonary hypertension comparable to smokers.

Systemic inflammation caused by white smoke inhalation in a combat exercise.

BACKGROUND: White smoke inhalation is an uncommon but potentially deadly cause of acute lung injury. No clinical spectrum or treatment protocol have been developed. METHODS: Twenty patients accidentally been exposed to white smoke during military training were the subjects of this study. We analyzed clinical manifestations, cytokine changes, and treatment outcomes. RESULTS: All patients initially presented with fever, dry cough, chest tightness, and shortness of breath. Twenty-five percent of these patients had severe acute lung injury requiring artificial ventilation support. Elevation of serum tumor necrosis factor-alpha was observed before treatment with antibiotics and glucocorticoids, but the elevation of transforming growth factor-beta(1) was delayed for 2 to 4 weeks after the accident. All the patients had leukocytosis, which correlated positively to disease severity and negatively to intensive treatments. Ninety-five percent of patients had varying degrees of restrictive ventilation impairment, and 85% of these patients had a significantly reduced diffusion capacity in the lungs. Seventy percent of these patients had transient impairment of liver function, which did not correlate to disease severity. The respiratory sequela of restrictive ventilation impairment developed in the most severely affected patients, whereas other tissue toxicities were mostly transient. Treatment included glucocorticoids, antibiotics, and respiratory therapy. All of the patients survived. CONCLUSION: A proper ventilation strategy, early pharmacologic therapy including glucocorticoids, and complication prevention may contribute to good treatment outcomes after white smoke inhalation.

Experimental exposure to wood-smoke particles in healthy humans: effects on markers of inflammation, coagulation, and lipid peroxidation.

Particulate air pollution is known to increase cardiovascular morbidity and mortality. Proposed mechanisms underlying this increase include effects on inflammation, coagulation factors, and oxidative stress, which could increase the risk of coronary events and atherosclerosis. The aim of this study was to examine whether short-term exposure to wood smoke affects markers of inflammation, blood hemostasis, and lipid peroxidation in healthy humans. Thirteen subjects were exposed to wood smoke and clean air in a chamber during two 4-h sessions, 1 wk apart. The mass concentrations of fine particles at wood smoke exposure were 240-280 mug/m3, and number concentrations were 95,000-180,000/cm3. About half of the particles were ultrafine (< 100 nm). Blood and urine samples were taken before and after the experiment. Exposure to wood smoke increased the levels of serum amyloid A, a cardiovascular risk factor, as well as factor VIII in plasma and the factor VIII/von Willebrand factor ratio, indicating a slight effect on the balance of coagulation factors. Moreover, there was an increased urinary excretion of free 8-iso-prostaglandin2alpha, a major F2-isoprostane, though this was based on nine subjects only, indicating a temporary increase in free radical-mediated lipid peroxidation. Thus, wood-smoke particles at levels that can be found in smoky indoor environments seem to affect inflammation, coagulation, and possibly lipid peroxidation. These factors may be involved in the mechanisms whereby particulate air pollution affects cardiovascular morbidity and mortality. The exposure setup could be used to establish which particle characteristics are critical for the effects.