Mucopolysaccharidosis (MPS IIIA) mice have increased lung compliance and airway resistance, decreased diaphragm strength, and no change in alveolar structure.

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterized by neurological and skeletal pathologies caused by reduced activity of the lysosomal hydrolase, sulfamidase, and the subsequent primary accumulation of undegraded heparan sulfate (HS). Respiratory pathology is considered secondary in MPS IIIA and the mechanisms are not well understood. Changes in the amount, metabolism, and function of pulmonary surfactant, the substance that regulates alveolar interfacial surface tension and modulates lung compliance and elastance, have been reported in MPS IIIA mice. Here we investigated changes in lung function in 20-wk-old control and MPS IIIA mice with a closed and open thoracic cage, diaphragm contractile properties, and potential parenchymal remodeling. MPS IIIA mice had increased compliance and airway resistance and reduced tissue damping and elastance compared with control mice. The chest wall impacted lung function as observed by an increase in airway resistance and a decrease in peripheral energy dissipation in the open compared with the closed thoracic cage state in MPS IIIA mice. Diaphragm contractile forces showed a decrease in peak twitch force, maximum specific force, and the force-frequency relationship but no change in muscle fiber cross-sectional area in MPS IIIA mice compared with control mice. Design-based stereology did not reveal any parenchymal remodeling or destruction of alveolar septa in the MPS IIIA mouse lung. In conclusion, the increased storage of HS which leads to biochemical and biophysical changes in pulmonary surfactant also affects lung and diaphragm function, but has no impact on lung or diaphragm structure at this stage of the disease.NEW & NOTEWORTHY Heparan sulfate storage in the lungs of mucopolysaccharidosis type IIIA (MPS IIIA) mice leads to changes in lung function consistent with those of an obstructive lung disease and includes an increase in lung compliance and airway resistance and a decrease in tissue elastance. In addition, diaphragm muscle contractile strength is reduced, potentially further contributing to lung function impairment. However, no changes in parenchymal lung structure were observed in mice at 20 wk of age.

Treatment with inhaled aerosolised ethanol reduces viral load and potentiates macrophage responses in an established influenza mouse model.

AIM: Treatment options for viral lung infections are currently limited. We aimed to explore the safety and efficacy of inhaled ethanol in an influenza-infection mouse model. MATERIALS AND METHODS: In a safety and tolerability experiment, 80 healthy female BALB/c mice (20 per group) were exposed to nebulized saline (control) or three concentrations of ethanol (40/60/80% ethanol v/v in water) for 3x30-minute periods, with a two-hour break between exposures. In a separate subsequent experiment, 40 Female BALB/c mice were nasally inoculated with 104.5 plaque-forming units of immediate virulence "Mem71" influenza. Infection was established for 48-h before commencing treatment in 4 groups of 10 mice with either nebulized saline (control) or one of 3 different concentrations of ethanol (40/60/80% ethanol v/v in water) for 3x30-minute periods daily over three consecutive days. In both experiments, mouse behavior, clinical scores, weight change, bronchoalveolar lavage cell viability, cellular composition, and cytokine levels, were assessed 24-h following the final exposure, with viral load also assessed after the second experiment. RESULTS: In uninfected BALB/c mice, 3x30-minute exposures to nebulized 40%, 60%, and 80% ethanol resulted in no significant differences in mouse weights, cell counts/viability, cytokines, or morphometry measures. In Mem71-influenza infected mice, we observed a dose-dependent reduction in viral load in the 80%-treated group and potentiation of macrophage numbers in the 60%- and 80%-treated groups, with no safety concerns. CONCLUSIONS: Our data provides support for inhaled ethanol as a candidate treatment for respiratory infections.

Respiratory Health Effects of In Vivo Sub-Chronic Diesel and Biodiesel Exhaust Exposure.

Biodiesel, which can be made from a variety of natural oils, is currently promoted as a sustainable, healthier replacement for commercial mineral diesel despite little experimental data supporting this. The aim of our research was to investigate the health impacts of exposure to exhaust generated by the combustion of diesel and two different biodiesels. Male BALB/c mice (n = 24 per group) were exposed for 2 h/day for 8 days to diluted exhaust from a diesel engine running on ultra-low sulfur diesel (ULSD) or Tallow or Canola biodiesel, with room air exposures used as control. A variety of respiratory-related end-point measurements were assessed, including lung function, responsiveness to methacholine, airway inflammation and cytokine response, and airway morphometry. Exposure to Tallow biodiesel exhaust resulted in the most significant health impacts compared to Air controls, including increased airway hyperresponsiveness and airway inflammation. In contrast, exposure to Canola biodiesel exhaust resulted in fewer negative health effects. Exposure to ULSD resulted in health impacts between those of the two biodiesels. The health effects of biodiesel exhaust exposure vary depending on the feedstock used to make the fuel.

Long-term exposure of mice to 890 ppm atmospheric CO2 alters growth trajectories and elicits hyperactive behaviours in young adulthood.

Atmospheric carbon dioxide (CO2 ) levels are currently at 418 parts per million (ppm), and by 2100 may exceed 900 ppm. The biological effects of lifetime exposure to CO2 at these levels is unknown. Previously we have shown that mouse lung function is altered by long-term exposure to 890 ppm CO2 . Here, we assess the broader systemic physiological responses to this exposure. Mice were exposed to either 460 or 890 ppm from preconception to 3 months of age, and assessed for effects on developmental, renal and osteological parameters. Locomotor, memory, learning and anxiety-like behaviours of the mice were also assessed. Exposure to 890 ppm CO2 increased birthweight, decreased female body weight after weaning, and, as young adults, resulted in reduced engagement in memory/learning tasks, and hyperactivity in both sexes in comparison to controls. There were no clear anxiety, learning or memory changes. Renal and osteological parameters were minimally affected. Overall, this study shows that exposure of mice to 890 ppm CO2 from preconception to young adulthood alters growth and some behaviours, with limited evidence of compensatory changes in acid-base balance. These findings highlight the potential for a direct effect of increased atmospheric CO2 on mammalian health outcomes. KEY POINTS: Long-term exposure to elevated levels of atmospheric CO2 is an uncontrolled experiment already underway. This is the first known study to assess non-respiratory physiological impacts of long-term (conception to young adulthood) exposure of mice to CO2 at levels that may arise in the atmosphere due to global emissions. Exposure to elevated CO2 , in comparison to control mice, altered growth patterns in early life and resulted in hyperactive behaviours in young adulthood. Renal and bone parameters, which are important to balance acid-base levels to compensate for increased CO2 exposure, remained relatively unaffected. This work adds to the body of evidence regarding the effects of carbon emissions on mammalian health and highlights a potential future burden of disease.

Azithromycin inhibits mucin secretion, mucous metaplasia, airway inflammation, and airways hyperresponsiveness in mice exposed to house dust mite extract.

Excessive production, secretion, and retention of abnormal mucus is a pathological feature of many obstructive airways diseases including asthma. Azithromycin is an antibiotic that also possesses immunomodulatory and mucoregulatory activities, which may contribute to the clinical effectiveness of azithromycin in asthma. The current study investigated these nonantibiotic activities of azithromycin in mice exposed daily to intranasal house dust mite (HDM) extract for 10 days. HDM-exposed mice exhibited airways hyperresponsiveness to aerosolized methacholine, a pronounced mixed eosinophilic and neutrophilic inflammatory response, increased airway smooth muscle (ASM) thickness, and elevated levels of epithelial mucin staining. Azithromycin (50 mg/kg sc, 2 h before each HDM exposure) attenuated HDM-induced airways hyperresponsiveness to methacholine, airways inflammation (bronchoalveolar lavage eosinophil and neutrophils numbers, and IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and RANTES levels), and epithelial mucin staining (mucous metaplasia) by at least 50% (compared with HDM-exposed mice, P < 0.05). Isolated tracheal segments of HDM-exposed mice secreted Muc5ac and Muc5b (above baseline levels) in response to exogenous ATP. Moreover, ATP-induced secretion of mucins was attenuated in segments obtained from azithromycin-treated, HDM-exposed mice (P < 0.05). In additional ex vivo studies, ATP-induced secretion of Muc5ac (but not muc5b) from HDM-exposed tracheal segments was inhibited by in vitro exposure to azithromycin. In vitro azithromycin also inhibited ATP-induced secretion of Muc5ac and Muc5b in tracheal segments from IL-13-exposed mice. In summary, azithromycin inhibited ATP-induced mucin secretion and airways inflammation in HDM-exposed mice, both of which are likely to contribute to suppression of airways hyperresponsiveness.

Previous Influenza Infection Exacerbates Allergen Specific Response and Impairs Airway Barrier Integrity in Pre-Sensitized Mice.

In this study we assessed the effects of antigen exposure in mice pre-sensitized with allergen following viral infection on changes in lung function, cellular responses and tight junction expression. Female BALB/c mice were sensitized to ovalbumin and infected with influenza A before receiving a second ovalbumin sensitization and challenge with saline, ovalbumin (OVA) or house dust mite (HDM). Fifteen days post-infection, bronchoalveolar inflammation, serum antibodies, responsiveness to methacholine and barrier integrity were assessed. There was no effect of infection alone on bronchoalveolar lavage cellular inflammation 15 days post-infection; however, OVA or HDM challenge resulted in increased bronchoalveolar inflammation dominated by eosinophils/neutrophils or neutrophils, respectively. Previously infected mice had higher serum OVA-specific IgE compared with uninfected mice. Mice previously infected, sensitized and challenged with OVA were most responsive to methacholine with respect to airway resistance, while HDM challenge caused significant increases in both tissue damping and tissue elastance regardless of previous infection status. Previous influenza infection was associated with decreased claudin-1 expression in all groups and decreased occludin expression in OVA or HDM-challenged mice. This study demonstrates the importance of the respiratory epithelium in pre-sensitized individuals, where influenza-infection-induced barrier disruption resulted in increased systemic OVA sensitization and downstream effects on lung function.

Electronic cigarettes: A position statement from the Thoracic Society of Australia and New Zealand.

The TSANZ develops position statements where insufficient data exist to write formal clinical guidelines. In 2018, the TSANZ addressed the question of potential benefits and health impacts of electronic cigarettes (EC). The working party included groups focused on health impacts, smoking cessation, youth issues and priority populations. The 2018 report on the Public Health Consequences of E-Cigarettes from the United States NASEM was accepted as reflective of evidence to mid-2017. A search for papers subsequently published in peer-reviewed journals was conducted in August 2018. A small number of robust and important papers published until March 2019 were also identified and included. Groups identified studies that extended, modified or contradicted the NASEM report. A total of 3793 papers were identified and reviewed, with summaries and draft position statements developed and presented to TSANZ membership in April 2019. After feedback from members and external reviewers, a collection of position statements was finalized in December 2019. EC have adverse lung effects and harmful effects of long-term use are unknown. EC are unsuitable consumer products for recreational use, part-substitution for smoking or long-term exclusive use by former smokers. Smokers who require support to quit smoking should be directed towards approved medication in conjunction with behavioural support as having the strongest evidence for efficacy and safety. No specific EC product can be recommended as effective and safe for smoking cessation. Smoking cessation claims in relation to EC should be assessed by established regulators.

Maternal high fat diet compromises survival and modulates lung development of offspring, and impairs lung function of dams (female mice).

BACKGROUND: Epidemiological studies have identified strong relationships between maternal obesity and offspring respiratory dysfunction; however, the causal direction is not known. We tested whether maternal obesity alters respiratory function of offspring in early life. METHODS: Female C57Bl/6 J mice were fed a high or low fat diet prior to and during two rounds of mating and resulting pregnancies with offspring lung function assessed at 2 weeks of age. The lung function of dams was measured at 33 weeks of age. RESULTS: A high fat diet caused significant weight gain prior to conception with dams exhibiting elevated fasting glucose, and glucose intolerance. The number of surviving litters was significantly less for dams fed a high fat diet, and surviving offspring weighed more, were longer and had larger lung volumes than those born to dams fed a low fat diet. The larger lung volumes significantly correlated in a linear fashion with body length. Pups born from the second pregnancy had reduced tissue elastance compared to pups born from the first pregnancy, regardless of the dam's diet. As there was reduced offspring survival born to dams fed a high fat diet, the statistical power of lung function measures of offspring was limited. There were signs of increased inflammation in the bronchoalveolar lavage fluid of dams (but not offspring) fed a high fat diet, with more tumour necrosis factor-α, interleukin(IL)-5, IL-33 and leptin detected. Dams that were fed a high fat diet and became pregnant twice had reduced fasting glucose immediately prior to the second mating, and lower levels of IL-33 and leptin in bronchoalveolar lavage fluid. CONCLUSIONS: While maternal high fat diet compromised litter survival, it also promoted somatic and lung growth (increased lung volume) in the offspring. Further studies are required to examine downstream effects of this enhanced lung volume on respiratory function in disease settings.

Soy Biodiesel Exhaust is More Toxic than Mineral Diesel Exhaust in Primary Human Airway Epithelial Cells.

As global biodiesel production increases, there are concerns over the potential health impact of exposure to the exhaust, particularly in regard to young children who are at high risk because of their continuing lung development. Using human airway epithelial cells obtained from young children, we compared the effects of exposure to exhaust generated by a diesel engine with Euro V/VI emission controls running on conventional diesel (ultra-low-sulfur mineral diesel, ULSD), soy biodiesel (B100), or a 20% blend of soy biodiesel with diesel (B20). The exhaust output of biodiesel was found to contain significantly more respiratory irritants, including NOx, CO, and CO2, and a larger overall particle mass. Exposure to biodiesel exhaust resulted in significantly greater cell death and a greater release of immune mediators compared to both air controls and ULSD exhaust. These results have concerning implications for potential global health impacts, particularly for the pediatric population.

Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells.

BACKGROUND: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. METHODS: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. RESULTS: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. CONCLUSIONS: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.

Foetal growth restriction in mice modifies postnatal airway responsiveness in an age and sex-dependent manner.

Epidemiological studies demonstrate an association between intrauterine growth restriction (IUGR) and asthma; however the underlying mechanism is unknown. We investigated the impact of maternal hypoxia-induced IUGR on airway responsiveness in male and female mice during juvenility and adulthood. Pregnant BALB/c mice were housed under hypoxic conditions for gestational days 11-17.5 and then returned to normoxic conditions for the remainder of pregnancy. A control group was housed under normoxic conditions throughout pregnancy. Offspring were studied at 2 weeks (juveniles) and 8 weeks (adults), where lung volume was assessed by plethysmography, airway responsiveness to methacholine determined by the forced oscillation technique and lungs fixed for morphometry. IUGR offspring were lighter at birth, exhibited "catch-up growth" by 2 weeks, but were again lighter in adulthood. IUGR males were "hyper-responsive" at 2 weeks and "hypo-responsive" as adults, in contrast with IUGR females who were hyper-responsive in adulthood. IUGR males had increased inner and total wall thickness at 2 weeks which resolved by adulthood, while airways in IUGR females were structurally normal throughout life. There were no differences in lung volume between Control and IUGR offspring at any age. Our data demonstrate changes in airway responsiveness as a result of IUGR that could influence susceptibility to asthma development and contribute to sexual dimorphism in asthma prevalence which switches from a male dominated disease in early life to a female dominated disease in adulthood.

Independent and combined effects of airway remodelling and allergy on airway responsiveness.

Airway remodelling and allergic inflammation are key features of airway hyperresponsiveness (AHR) in asthma; however, their interrelationships are unclear. The present study investigated the separate and combined effects of increased airway smooth muscle (ASM) layer thickness and allergy on AHR. We integrated a protocol of ovalbumin (OVA)-induced allergy into a non-inflammatory mouse model of ASM remodelling induced by conditional and airway-specific expression of transforming growth factor-α (TGF-α) in early growth response-1 (Egr-1)-deficient transgenic mice, which produced thickening of the ASM layer following ingestion of doxycycline. Mice were sensitised to OVA and assigned to one of four treatment groups: Allergy - normal chow diet and OVA challenge; Remodelling - doxycycline in chow and saline challenge; Allergy and Remodelling - doxycycline in chow and OVA challenge; and Control - normal chow diet and saline challenge. Airway responsiveness to methacholine (MCh) and histology were assessed. Compared with the Control group, airway responsiveness to MCh was increased in the Allergy group, independent of changes in wall structure, whereas airway responsiveness in the Remodelling group was increased independent of exposure to aeroallergen. The combined effects of allergy and remodelling on airway responsiveness were greater than either of them alone. There was a positive relationship between the thickness of the ASM layer with airway responsiveness, which was shifted upward in the presence of allergy. These findings support allergy and airway remodelling as independent causes of variable and excessive airway narrowing.

The effects of electronic cigarette aerosol exposure on inflammation and lung function in mice.

Electronic cigarette usage is increasing worldwide, yet there is a paucity of information on the respiratory health effects of electronic cigarette aerosol exposure. This study aimed to assess whether exposure to electronic cigarette (e-cigarette) aerosol would alter lung function and pulmonary inflammation in mice and to compare the severity of any alterations with mice exposed to mainstream tobacco smoke. Female BALB/c mice were exposed for 8 wk to tobacco smoke, medical air (control), or one of four different types of e-cigarette aerosol. E-cigarette aerosols varied depending on nicotine content (0 or 12 mg/ml) and the main excipient (propylene glycol or glycerin). Twenty-four hours after the final exposure, we measured pulmonary inflammation, lung volume, lung mechanics, and responsiveness to methacholine. Mice exposed to tobacco cigarette smoke had increased pulmonary inflammation and responsiveness to methacholine compared with air controls. Mice exposed to e-cigarette aerosol did not have increased inflammation but did display decrements in parenchymal lung function at both functional residual capacity and high transrespiratory pressures. Mice exposed to glycerin-based e-cigarette aerosols were also hyperresponsive to methacholine regardless of the presence or absence of nicotine. This study shows, for the first time, that exposure to e-cigarette aerosol during adolescence and early adulthood is not harmless to the lungs and can result in significant impairments in lung function.

Increased heterogeneity of airway calibre in adult rats after hypoxia-induced intrauterine growth restriction.

BACKGROUND AND OBJECTIVE: Intrauterine growth restriction (IUGR) is associated with asthma development. We hypothesized that IUGR disrupts airway development leading to postnatal structural abnormalities of the airway that predispose to disease. This study therefore examined structural changes to the airway and lung in a rat model of maternal hypoxia-induced IUGR. METHODS: Pregnant rats were housed under hypoxic conditions (11.5% O2 ) from gestational days (GDs) 13 to 20 (pseudoglandular-canalicular stages, i.e. period of airway development) and then returned to normoxic conditions (21% O2 ). A control group of pregnant rats was housed under normoxic conditions throughout pregnancy. Weights of male offspring were recorded at birth and 7 weeks of age (adulthood), at which point lungs were fixed for morphometry and stereology (n = 6/group), or bronchoalveolar lavage fluid (BALF) was collected for cell counts (n = 6/group). RESULTS: IUGR offspring were lighter at birth compared with control, but not at 7 weeks. While there was no difference in mean airway dimensions or lung volume, there was greater anatomical variation in airway lumen area in the IUGR group. A mathematical model of the human lung was used to show that greater heterogeneity in lumen area in IUGR-affected individuals increases bronchoconstriction during simulated bronchial challenge. More macrophages were identified in the BALF of IUGR offspring. CONCLUSION: The rat model demonstrates that IUGR leads to a more heterogeneous distribution of airway lumen calibre in adulthood with potential implications for bronchoconstriction in human subjects. Together with increased lung macrophages, these findings support a phenotypic shift after IUGR that may impact disease susceptibility.

Early life rhinovirus infection exacerbates house-dust-mite induced lung disease more severely in female mice.

PURPOSE: Recent studies have employed animal models to investigate links between rhinovirus infection and allergic airways disease, however, most do not involve early life infection, and none consider the effects of sex on responses. MATERIALS AND METHODS: Here, we infected male and female mice with human rhinovirus 1B (or control) on day 7 of life. Mice were then subjected to 7 weeks of exposure to house-dust-mite prior to assessment of bronchoalveolar inflammation, serum antibodies, lung function, and responsiveness to methacholine. RESULTS: There were significant differences in responses between males and females in most outcomes. In males, chronic house-dust-mite exposure increased bronchoalveolar inflammation, house-dust-mite specific IgG1 and responsiveness of the lung parenchyma, however, there was no additional impact of rhinovirus infection. Conversely, in females, there were additive and synergistic effects of rhinovirus infection and house-dust-mite exposure on neutrophilia, airway resistance, and responsiveness of the lung parenchyma. CONCLUSIONS: We conclude that early life rhinovirus infection influences the development of house-dust-mite induced lung disease in female, but not male mice.

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.

RATIONALE: No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. AIM OF THE STUDY: To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. MATERIALS AND METHODS: Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID50) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. RESULTS: HRV-1B infection affected viability that was both time and TCID50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID50, while a significant decrease in all three TJ protein expressions occurred at higher TCID50. Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. CONCLUSION: HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

Biodiesel exhaust-induced cytotoxicity and proinflammatory mediator production in human airway epithelial cells.

Increasing use of biodiesel has prompted research into the potential health effects of biodiesel exhaust exposure. Few studies directly compare the health consequences of mineral diesel, biodiesel, or blend exhaust exposures. Here, we exposed human epithelial cell cultures to diluted exhaust generated by the combustion of Australian ultralow-sulfur-diesel (ULSD), unprocessed canola oil, 100% canola biodiesel (B100), and a blend of 20% canola biodiesel mixed with 80% ULSD. The physicochemical characteristics of the exhaust were assessed and we compared cellular viability, apoptosis, and levels of interleukin (IL)-6, IL-8, and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) in exposed cultured cells. Different fuel types produced significantly different amounts of exhaust gases and different particle characteristics. All exposures resulted in significant apoptosis and loss of viability when compared with control, with an increasing proportion of biodiesel being correlated with a decrease in viability. In most cases, exposure to exhaust resulted in an increase in mediator production, with the greatest increases most often in response to B100. Exposure to pure canola oil (PCO) exhaust did not increase mediator production, but resulted in a significant decrease in IL-8 and RANTES in some cases. Our results show that canola biodiesel exhaust exposure elicits inflammation and reduces viability of human epithelial cell cultures in vitro when compared with ULSD exhaust exposure. This may be related to an increase in particle surface area and number in B100 exhaust when compared with ULSD exhaust. Exposure to PCO exhaust elicited the greatest loss of cellular viability, but virtually no inflammatory response, likely due to an overall increase in average particle size.