Master of Science in Experimental Medicine (MSc)
Identifying a Biosignature of Occupational Diesel Exhaust Exposure by Studying Dose-response in a Controlled System
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Phthalates are used as softeners in commercial products. They leak into the environment and become wide-spread contaminants. Epidemiological studies suggest an association between phthalate inhalation and development/worsening of airway diseases, but a firm link has not been established. Asthma is a complex disorder associated with inflammation resulting in airway hyper-responsiveness (AHR). Increasing interest is focused on abnormal immune responses as an underlying mechanism contributing to increasing the risk of asthma.This study is the first to investigate airway and systemic effects in humans due to inhalation of a known concentration of a single phthalate. Di-butyl phthalate (DBP), exists in high concentrations in indoor air and has shown inflammatory potential. We hypothesize that DBP inhalation, prior to allergen inhalation will: a) enhance airway inflammation and responsiveness to allergen, and b) alter the activation state and functionality of immune cells in the airway and peripheral blood.In this novel double-blind, order-randomized, crossover study, 16 participants were exposed by inhalation to controlled levels of DBP or clean air (CA) for 3h, followed immediately by an allergen (dust-mite, grass or birch) inhalation. To assess lung function and airway inflammation, spirometry and exhaled nitric oxide was measured before, 3h and 20h post-DBP/CA exposure and allergen inhalation. Blood, bronchoalveolar wash and lavage (BAL) were collected for quantification and measurement of the activation pattern of immune cells and inflammatory mediator release.DBP inhalation followed by an allergen inhalation, significantly augmented the airflow decline (FEV1) in response to an inhaled allergen, compared to CA. Moreover, DBP enhanced the recruitment of BAL macrophages, specifically the M2 phenotype with increased expression of CD206 to the lungs. Meanwhile, the percent of T helper cells increased, while T regulatory and non-classical monocytes decreased, in peripheral blood. Only minor effects were observed for systemic inflammatory mediators. Moreover, significant effect modifications were observed for sex, AHR status and type of allergen inhaled.The results suggest significant effects of a common commercial chemical, DBP, on clinically relevant airway and systemic endpoints in the context of allergen exposure in sensitized individuals. Future research should aim to validate and connect these findings within relevant policy and public health contexts.
Allergic rhinitis is a global health problem that causes major illness and disability. Inherited and environmental factors influence its development. This thesis examined the role of traffic-related air pollution, genetic variants and their potential interactions, on childhood allergic rhinitis. Global spatial associations with climatic factors known to influence aeroallergen distributions were also studied. Data from two Canadian (CAPPS and SAGE) and four European birth cohorts (BAMSE, GINIplus, LISAplus and PIAMA) participating in the Traffic, Asthma and Genetics collaboration were pooled. No consistent associations between individual-level traffic-related air pollutants (NO2, PM2.5 mass, PM2.5 absorbance and ozone) estimated to the home address and childhood allergic rhinitis were observed in a longitudinal analysis (up to ten years) of two cohorts (GINIplus and LISAplus; N=6,604) and a pooled analysis of all six cohorts (N=15,299). These latter null associations were not modified by ten tested single nucleotide polymorphisms in the GSTP1, TNF, TLR2 and TLR4 genes. Although these results do not support an adverse role of traffic-related air pollution on childhood allergic rhinitis, much remains to be learned regarding for whom, when and how air pollution may impact disease.In further analyses, genetic variants in the TNF and TLR4 genes and at the 17q21 gene locus were found to be associated with childhood allergic rhinitis in pooled analyses of the six cohorts. As genetic variability in these regions has also been linked to asthma, the observed associations support the hypothesis of shared genetic susceptibility between asthma and allergic rhinitis. These results may be important for public health given the large proportion of the population carrying the studied risk variants.Lastly, using cross-sectional data from 6-7 and 13-14 year-olds participating in the International Study of Asthma and Allergies in Childhood, several ecological spatial associations between climatic factors (temperature, precipitation and vapour pressure) and intermittent and persistent rhinitis symptom prevalences were identified. Although not conclusive, these results represent a first step in investigating how climate change may affect rhinitis symptom prevalence.Collectively, this dissertation contributes to our understanding of the effects of air pollution, genetic variability and climate on childhood allergic rhinitis.
Introduction: Traffic-related air pollution (TRAP) is associated with COPD epidemiologically, however the mechanism of this interaction remains unclear. Neutrophils, a key inflammatory cell in COPD, migrate to the lungs following TRAP exposure, but their functional role in this response is poorly understood. The aim of this study is to elucidate the effects of TRAP exposure on neutrophil function, using the model of diesel exhaust (DE).Methods: In vitro: Isolated peripheral blood neutrophils were incubated with diesel exhaust particles (DEPs) before quantifying activation marker expression, oxidative burst, and neutrophil extracellular traps (NETs). In vivo: Subjects from three groups (never-smokers, ex-smokers, and mild-moderate COPD) participated in a randomized double-blind controlled human exposure crossover study. Subjects were exposed on one occasion to filtered air and on another occasion to diluted DE. Blood and bronchoalveolar lavage (BAL) samples were used to assess the effect of DE on the number and function of pulmonary and systemic neutrophils.Results: DEPs increased CD66b, oxidative burst, and NET formation in vitro. Controlled human exposure to DE reduced the proportion of circulating band cells, increased NET formation in BAL, and resulted in lymphocytic but not neutrophilic inflammation. The effect of DE on band cells and peripheral CD182 expression was distinct in the COPD group. There was no effect on oxidative burst or activation marker expression in pulmonary neutrophils.Conclusion: Diesel exhaust increases some, but not all, neutrophil effector activities in vitro and in vivo. Two potential mechanisms for susceptibility in COPD patients were identified. These results demonstrate a functional role for neutrophils in the inflammatory response to diesel exhaust. The potential for DE-induced neutrophil activity to promote lung tissue damage and clinical features of COPD may be an area of future investigation.
Background: Diesel exhaust (DE) is a common exposure in Canadian workplaces. The International Agency for Research on Cancer (IARC) classified DE as being carcinogenic to humans in 2012. Health and safety agencies provide information about DE and the mitigation strategies that can be used to reduce the exposure of individuals. However, there is little known about the extent to which those potentially exposed in the workplace understand the risks of DE or have recently changed behaviours to minimize workplace exposure to DE. Objectives: To identify exposure-related knowledge, attitudes and behaviours of individuals occupationally exposed to diesel exhaust; to reveal strengths, knowledge gaps and misperceptions therein. Methods: A Mental Models approach was used to gather information about current scientific understanding of DE exposure hazards and the ways in which exposure can be reduced. Thirty individuals in British Columbia who were regularly exposed to occupational DE were interviewed. The audio was recorded, transcribed, grouped together, and examined to draw out themes around DE awareness, hazard assessment and risk reduction behaviours. These themes were then compared and contrasted with existing grey and research literature in order to reveal strengths, gaps and misperceptions regarding exposure to DE. Results: Study participants were aware and concerned about DE but had incomplete and sometimes incorrect understanding of exposure pathways, health effects, and effective strategies to reduce their exposures. The perceived likelihood of exposure to diesel exhaust was significantly greater compared to that of other work hazards (p
Asthma is a chronic condition described by inflammation of the airways. There are no specific treatments for asthma yet but understanding the potential triggers of an asthma attack can effectively control this disease and make it more manageable for many sufferers. Therefore, there is intense interest in studying the negative impacts of air pollutants on respiratory diseases. Diesel exhaust (DE) is a primary source of emissions from motor vehicles and is also a significant cause of increased airway responsiveness in asthma. The main particulate fraction of diesel exhaust consists of fine particles (PM₂․₅), which are inhaled efficiently into the lung. Inhalation of these particles can exacerbate asthma and trigger other harmful processes in the lung. The effect of traffic-related air pollution, such as DE on asthma exacerbations is well-established but the biological mechanism underlying this association is still not well understood. DE is thought to interact with allergen exposures to mediate adverse effects, but most of the studies done in this area are based on animal models and there remains poor appreciation of the mechanisms of allergen-DE synergy in human models. In this research project, we aim to elucidate if DE increases bronchial allergen-induced inflammation and cellular immune response in mild atopic asthmatic human subjects. Volunteer participants were exposed to DE (300 μg.m-³ of PM₂․₅) or filtered air for two hours in a blinded crossover study design with a four-week washout period. One hour following either filtered air or DE exposure, subjects were exposed to allergen or saline via bronchoscopic segmental challenge. Forty-eight hours post-exposure, endobronchial biopsies were collected. Tissue sections were immunostained for tryptase, eosinophil cationic protein (ECP), neutrophil elastase (NE), CD138, CD4 and interleukin (IL)-4. The percent positivity of positive cells were quantified in the bronchial submucosa by Aperio ImageScope Software. We have shown that in vivo allergen and DE co-exposure results in elevated CD4, IL-4, CD138 and NE in the respiratory submucosa of atopic subjects, while eosinophils and mast cells are not changed. Here we demonstrated, for the first time, the effect of DE exposure in promoting allergen-induced inflammatory responses directly within the lungs of atopic human.
Epidemiological and animal studies suggest that exposure to airborne pollutants may negatively impact the central nervous system (CNS). It is thought that traffic related air pollution (TRAP), and other forms of combustion-derived pollutants, may induce a maladaptive activation of the CNS immune system, however, the exact pathway is not understood. Animal models and epidemiological studies have inherent limitations including potential interspecies differences and residual confounding. Given this, the aim of this research is to examine effects of TRAP on the CNS using a controlled human exposure. 27 healthy adults were exposed to two conditions: filtered air (FA) and diesel exhaust (DE) (300µg PM₂.₅/m³) for 120 minutes, in a double-blinded crossover study with exposures separated by four-weeks. Prior to and at 0, 3, and 24 hours following exposure, serum and plasma were collected and analyzed for inflammatory cytokines IL-6 and TNF-α, the astrocytic protein S100b, the neuronal cytoplasmic enzyme neuron specific enolase (NSE), and brain derived neurotrophic factor (BDNF). The hypothesis was that IL-6, TNF-α, S100b and NSE would increase and BDNF would decrease following DE exposure. Changes in levels of biomarkers were assessed using a paired t-test to compare the change from baseline at each post-exposure timepoint following DE or FA exposure. A linear mixed effects model was build including exposure and timepoint as covariates, and subject ID as a random effect. Age and gender were examined as potential effect-modifying variables. At no time-point following exposure to DE was a significant increase from baseline seen for IL-6, TNF-α, S100b or NSE, or decrease for BDNF, relative to FA exposure. The linear mixed effects model revealed indication of diurnal behavior for S100B, NSE and BDNF; however, no significant exposure-time-point interaction, suggesting the biomarkers were not affected by DE exposure. These results indicate that short-term exposure to DE amongst young, healthy adults does not acutely affect levels of the measured biomarkers. This study does not disprove a relationship between air pollution and adverse CNS effects and suggests a need to examine the effects of TRAP on the brain using in chronic exposure models or more sensitive CNS endpoints.
Introduction: Adipokines are inflammatory mediators released primarily from the adipose tissue. These proteins are now recognized as active elements of systemic and pulmonary inflammatory responses, whose dysregulation can prime the development of allergic lung diseases. The purpose of this study was to measure adipokine responses in an atopic adult study population following exposure to allergen and diesel exhaust. Characterizing adipokine responses in the lung and in the serum, in an atopic but otherwise healthy population, can provide insight into adipokine responses in sensitized to specific allergens, yet without co-morbidities. Methods: Lung and blood samples were collected from subjects participating in a randomized, double-blinded controlled human study with crossover to two conditions: inhaled diesel exhaust and inhaled filtered air, each of which were followed by lung-instilled allergen (and contralateral saline control). Serum samples collected at baseline, 4, 24 and 48 hours after allergen instillation, and lung samples collected at 48 hours after allergen were assayed for total adiponectin, leptin and resistin using ELISA. Mixed-effects models were used for statistical analysis to determine exposure effect, and effect modification by sex, BMI status and airway responsiveness. Results: Adiponectin and leptin were significantly increased in the lung in response to allergen. Leptin and resistin changed in the serum in a diurnal pattern, but levels were not altered by diesel exhaust. Diesel exhaust and allergen co-exposure significantly increased the adiponectin/leptin ratio in the lung relative to allergen alone, in subjects with normal airway responsiveness. Conclusion: Increases in lung adipokines in response to allergen exposure were identified in the context of a controlled human exposure study. Some effect modification by sex, BMI and airway responsiveness occurred. Diesel exhaust along with allergen induced a protective adipokine pattern in the lung in those with normal airway responsiveness. The clinical relevance and generalizability of these findings, herein noted in atopic individuals, warrants further study.