Christopher Carlsten

While allergen inhalation has been a primary focus of asthma research, the evidence associating traffic-related air pollution (TRAP) with allergic asthma is growing, but the underlying interactions remain unclear. The airway epithelium is amongst the first cells exposed to TRAP, hence understanding its interactions with TRAP and allergen is important. Diesel exhaust (DE), a paradigm of TRAP, consists of particulate matter (PM) and gases. Modern diesel engines often have catalytic diesel particulate filters (CDPF) to reduce PM output, but these may increase gas release, and its effects on human health remain unclear. We conducted a randomized, double-blinded, crossover study using our unique in vivo human exposure system to investigate the effects of DE and allergen co-exposure, with or without particle depletion to model CDPF, on the airway epithelial transcriptome. Participants were exposed for 2 hours before an allergen inhalation challenge, with each receiving filtered air and saline (FA-S), filtered air and allergen (FA-A), DE and allergen (DE-A), or particle-depleted DE and allergen (PDDE-A), over 4 different occasions, each separated by a 4-week washout period. Endobronchial brushings were collected 48 hours after each exposure and RNA sequenced. Quality was checked and alignment and quantification against a reference transcriptome performed. Differentially expressed genes (DEGs) were identified using DESeq2 followed by GO enrichment and pathway analysis. FA-A, DE-A, and PDDE-A exposures significantly modulated genes relative to FA-S. Therein, 462 DEGs were identified with 6 (↑4, ↓2) modulated by all three conditions; FA-A uniquely modulated the highest number (↑178, ↓155), followed by DE-A (↑44, ↓23), and then PDDE-A exposure (↑15, ↓2). Investigation revealed that FA-A and DE-A exposures modulated gene expression associated with inflammatory responses and that some of these were reduced after particle depletion. Taken together, this thesis provides experimental evidence and novel insights in elucidating transcriptional changes after co-exposure to DE and allergens, with or without particle depletion.

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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.

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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
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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.

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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.

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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.

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Introduction: The purpose of this study was to characterize the effects of a controlled diesel exhaust (DE) exposure on airway oxidative stress in humans by measuring two biomarkers of interest in exhaled breath condensate (EBC). As there is evidence that antioxidant supplementation plays a role in reducing respiratory health effects associated with DE, this study also assessed whether antioxidant supplementation helps mitigate DE-related oxidative stress in humans.Methods: EBC was taken from subjects participating in a randomized, three-way crossover study (i.e. 3 different exposures: fresh air + placebo [FAP], DE + placebo [DEP], and DE + antioxidant [DEN]) at baseline as well as 2, 6 and 30 hours after exposure. Analysis for 8-isoprostane was performed using liquid chromatography with tandem mass spectrometry; pH was analyzed using standard de-aeration protocol and pH meter reading. Linear mixed effects models in SPLUS 8.0 were used for statistical analysis. Results: A total of 27 participants had their EBC collected and analysed for biomarker content: 23 for 8-isoprostane and 17 for pH. 8-isoprostane was consistently higher after DEP relative to FAP and was consistently lower after DEN relative to DEP, but none of these trends were statistically significant. Effects of exposure on pH were less consistent. The effect of exposure on 6 hour pH was significantly modified by sex (p=0.03); males showed a significant acidification after DEP relative to FAP (p=0.003), females showed a significant acidification after DEP relative to DEN (p=0.03). Other covariates did not significantly modify the interaction between exposure and biomarker levels. Conclusion: Amongst all subjects, exposure had no significant effect on EBC oxidative stress biomarkers. According to a secondary analysis, DEP lowered EBC pH 6 hours after exposure in males. Short-term diesel exhaust at concentrations typical of occupational settings does not significantly alter EBC oxidative stress in a controlled study with modest sample size. However, trends towards an effect on pH and apparent effect modification by gender warrant consideration of further study using a larger sample size.

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