TOLLIP and MUC5B modulate the effect of ambient NO2 on respiratory symptoms in infancy.

BACKGROUND: Current knowledge suggests that the gene region containing MUC5B and TOLLIP plays a role in airway defence and airway inflammation, and hence respiratory disease. It is also known that exposure to air pollution increases susceptibility to respiratory disease. We aimed to study whether the effect of air pollutants on the immune response and respiratory symptoms in infants may be modified by polymorphisms in MUC5B and TOLLIP genes. METHODS: 359 healthy term infants from the prospective Basel-Bern Infant Lung Development (BILD) birth cohort were included in the study. The main outcome was the score of weekly assessed respiratory symptoms in the first year of life. Using the candidate gene approach, we selected 10 single nucleotide polymorphisms (SNPs) from the MUC5B and TOLLIP regions. Nitrogen dioxide (NO2) and particulate matter ≤10 µm in aerodynamic diameter (PM10) exposure was estimated on a weekly basis. We used generalised additive mixed models adjusted for known covariates. To validate our results in vitro, cells from a lung epithelial cell line were downregulated in TOLLIP expression and exposed to diesel particulate matter (DPM) and polyinosinic-polycytidylic acid. RESULTS: Significant interaction was observed between modelled air pollution (weekly NO2 exposure) and 5 SNPs within MUC5B and TOLLIP genes regarding respiratory symptoms as outcome: E.g., infants carrying minor alleles of rs5744034, rs3793965 and rs3750920 (all TOLLIP) had an increased risk of respiratory symptoms with increasing NO2 exposure. In vitro experiments showed that cells downregulated for TOLLIP react differently to environmental pollutant exposure with DPM and viral stimulation. CONCLUSION: Our findings suggest that the effect of air pollution on respiratory symptoms in infancy may be influenced by the genotype of specific SNPs from the MUC5B and TOLLIP regions. For validation of the findings, we provided in vitro evidence for the interaction of TOLLIP with air pollution.

Differences in autophagy marker levels at birth in preterm vs. term infants.

BACKGROUND: Preterm infants are susceptible to oxidative stress and prone to respiratory diseases. Autophagy is an important defense mechanism against oxidative-stress-induced cell damage and involved in lung development and respiratory morbidity. We hypothesized that autophagy marker levels differ between preterm and term infants. METHODS: In the prospective Basel-Bern Infant Lung Development (BILD) birth cohort we compared cord blood levels of macroautophagy (Beclin-1, LC3B), selective autophagy (p62) and regulation of autophagy (SIRT1) in 64 preterm and 453 term infants. RESULTS: Beclin-1 and LC3B did not differ between preterm and term infants. However, p62 was higher (0.37, 95% confidence interval (CI) 0.05;0.69 in log2-transformed level, p = 0.025, padj = 0.050) and SIRT1 lower in preterm infants (-0.55, 95% CI -0.78;-0.31 in log2-transformed level, padj < 0.001). Furthermore, p62 decreased (padj-value for smoothing function was 0.018) and SIRT1 increased (0.10, 95% CI 0.07;0.13 in log2-transformed level, padj < 0.001) with increasing gestational age. CONCLUSION: Our findings suggest differential levels of key autophagy markers between preterm and term infants. This adds to the knowledge of the sparsely studied field of autophagy mechanisms in preterm infants and might be linked to impaired oxidative stress response, preterm birth, impaired lung development and higher susceptibility to respiratory morbidity in preterm infants. IMPACT: To the best of our knowledge, this is the first study to investigate autophagy marker levels between human preterm and term infants in a large population-based sample in cord blood plasma This study demonstrates differential levels of key autophagy markers in preterm compared to term infants and an association with gestational age This may be linked to impaired oxidative stress response or developmental aspects and provide bases for future studies investigating the association with respiratory morbidity.

Diagnostic testing in people with primary ciliary dyskinesia: An international participatory study.

Diagnostic tests are important in primary ciliary dyskinesia (PCD), a rare disease, to confirm the diagnosis and characterize the disease. We compared diagnostic tests for PCD between countries worldwide, assessed whether people with PCD recall their tests, and identified factors associated with the use of tests. We used cross-sectional data from COVID-PCD-an international participatory cohort study collecting information directly from people with PCD. The baseline questionnaire inquired about tests used for PCD diagnosis. Using logistic regression, we investigated factors associated with measurement of nasal nitric oxide (nNO), biopsy for electron or video microscopy, and genetic testing. We included data from 747 participants (60% females) from 49 countries worldwide with median age 27 (interquartile range 12-44). Most (92%) reported diagnostic tests for PCD. Participants reported measurements of nNO (342; 49%), biopsy samples (561; 75%), and genetic tests (435; 58%). The reported use of individual tests, such as genetics, varied between countries from 38% in Switzerland to 68% in North America. Participant recall of test type also differed between countries with lowest recall in Switzerland. One-third (232; 36%) of participants reported all three tests (nNO, biopsy, and genetics). Recently diagnosed people reported more tests [nNO odds ratio (OR) 2.2, 95% Confidence Interval (CI) 1.5-3.2; biopsy OR 3.2, 95%CI 2.1-4.9; genetics OR 4.7, 95%CI 3.2-6.9] and those with situs abnormalities fewer tests (nNO OR 0.5, 95%CI 0.4-0.7; biopsy OR 0.5, 95%CI 0.4-0.8; genetics OR 0.7, 95%CI 0.5-0.94). Our results indicate PCD diagnostic testing differed widely around the world and many patients received incomplete diagnostic work-up based only on clinical features or single tests. People diagnosed long ago and those with situs abnormalities possibly benefit from supplementary testing to refine their diagnosis as a prerequisite for personalized medicine.

The Cilialyzer - A freely available open-source software for the analysis of mucociliary activity in respiratory cells.

BACKGROUND AND OBJECTIVE: Primary ciliary dyskinesia (PCD) is a rare genetic disorder causing a defective ciliary structure, which predominantly leads to an impaired mucociliary clearance and associated airway disease. As there is currently no single diagnostic gold standard test, PCD is diagnosed by a combination of several methods comprising genetic testing and the examination of the ciliary structure and function. Among the approved diagnostic methods, only high-speed video microscopy (HSVM) allows to directly observe the ciliary motion and therefore, to directly assess ciliary function. In the present work, we present our recently developed freely available open-source software - termed "Cilialyzer", which has been specifically designed to support and facilitate the analysis of the mucociliary activity in respiratory epithelial cells captured by high-speed video microscopy. METHODS: In its current state, the Cilialyzer software enables clinical PCD analysts to load, preprocess and replay recorded image sequences as well as videos with a feature-rich replaying module facilitating the commonly performed qualitative visual assessment of ciliary function (including the assessment of the ciliary beat pattern). The image processing methods made accessible through an intuitive user interface allow clinical specialists to comfortably compute the ciliary beating frequency (CBF), the activity map and the "frequency correlation length" - an observable getting newly introduced. Furthermore, the Cilialyzer contains a simple-to-use particle tracking interface to determine the mucociliary transport speed. RESULTS: Cilialyzer is fully written in the Python programming language and freely available under the terms of the MIT license. The proper functioning of the computational analysis methods constituting the Cilialyzer software is demonstrated by using simulated and representative sample data from clinical practice. Additionally, the software was used to analyze high-speed videos showing samples obtained from healthy controls and genetically confirmed PCD cases (DNAI1 and DNAH11 mutations) to show its clinical applicability. CONCLUSIONS: Cilialyzer serves as a useful clinical tool for PCD analysts and provides new quantitative information awaiting to be clinically evaluated using cohorts of PCD. As Cilialyzer is freely available under the terms of a permissive open-source license, it serves as a ground frame for further development of computational methods aiming at the quantification and automation of the analysis of mucociliary activity captured by HSVM.

In Vitro Effect of Combined Hypertonic Saline and Salbutamol on Ciliary Beating Frequency and Mucociliary Transport in Human Nasal Epithelial Cells of Healthy Volunteers and Patients with Cystic Fibrosis.

Background: Inhalation of hypertonic saline (HS) is standard of care in patients with cystic fibrosis (CF). However, it is unclear if adding salbutamol has-besides bronchodilation-further benefits, for example, on the mucociliary clearance. We assessed this in vitro by measuring the ciliary beating frequency (CBF) and the mucociliary transport rate (MCT) in nasal epithelial cells (NECs) of healthy volunteers and patients with CF. Aims: To investigate the effect of HS, salbutamol, and its combination on (muco)ciliary activity of NECs in vitro, and to assess potential differences between healthy controls and patients with CF. Methods: NECs obtained from 10 healthy volunteers and 5 patients with CF were differentiated at the air-liquid interface and aerosolized with 0.9% isotonic saline ([IS] control), 6% HS, 0.06% salbutamol, or combined HS and salbutamol. CBF and MCT were monitored over 48-72 hours. Results: In NECs of healthy controls, the absolute CBF increase was comparable for all substances, but CBF dynamics were different: HS increased CBF slowly and its effect lasted for an extended period, salbutamol and IS increased CBF rapidly and the effect subsided similarly fast, and HS and salbutamol resulted in a rapid and long-lasting CBF increase. Results for CF cells were comparable, but less pronounced. Similar to CBF, MCT increased after the application of all the tested substances. Conclusion: CBF and MCT of NECs of healthy participants and CBF of patients with CF increased upon treatment with aerosolized IS, HS, salbutamol, or HS and salbutamol, showing a relevant effect for all tested substances. The difference in the CBF dynamics can be explained by the fact that the properties of the mucus are changed differently by different saline concentrations.

How I Do It: Transperineal prostate biopsy using local anesthetic in an outpatient setting.

Transperineal prostate biopsy (TPPB) is proven to be an effective diagnostic tool for prostate cancer detection. It allows satisfactory sampling of apical and anterior areas which is not well achieved with the transrectal route, without the associated risks of urinary tract infection or sepsis. The main objective of this paper is to describe the technique utilized in our institution to perform transperineal prostate biopsy under local anesthetic in the outpatient clinic setting.

Respiratory symptoms of Swiss people with primary ciliary dyskinesia.

Background: Mostly derived from chart reviews, where symptoms are recorded in a nonstandardised manner, clinical data about primary ciliary dyskinesia (PCD) are inconsistent, which leads to missing and unreliable information. We assessed the prevalence and frequency of respiratory and ear symptoms and studied differences by age and sex among an unselected population of Swiss people with PCD. Methods: We sent a questionnaire that included items from the FOLLOW-PCD standardised questionnaire to all Swiss PCD registry participants. Results: We received questionnaires from 74 (86%) out of 86 invited persons or their caregivers (median age 23 years, range 3-73 years), including 68% adults (≥18 years) and 51% females. Among participants, 70 (94%) reported chronic nasal symptoms; most frequently runny nose (65%), blocked nose (55%) or anosmia (38%). Ear pain and hearing problems were reported by 58% of the participants. Almost all (99%) reported cough and sputum production. The most common chronic cough complications were gastro-oesophageal reflux (n=11; 15%), vomiting (n=8; 11%) and urinary incontinence (n=6; 8%). Only nine (12%) participants reported frequent wheeze, which occurred mainly during infection or exercise, while 49 (66%) reported shortness of breath, and 9% even at rest or during daily activities. Older patients reported more frequent nasal symptoms and shortness of breath. We found no difference by sex or ultrastructural ciliary defect. Conclusion: This is the first study to describe patient-reported PCD symptoms. The consistent collection of standardised clinical data will allow us to better characterise the phenotypic variability of the disease and study disease course and prognosis.

Diagnosis of primary ciliary dyskinesia: discrepancy according to different algorithms.

BACKGROUND: Diagnosis of primary ciliary dyskinesia (PCD) is challenging since there is no gold standard test. The European Respiratory (ERS) and American Thoracic (ATS) Societies developed evidence-based diagnostic guidelines with considerable differences. OBJECTIVE: We aimed to compare the algorithms published by the ERS and the ATS with each other and with our own PCD-UNIBE algorithm in a clinical setting. Our algorithm is similar to the ERS algorithm with additional immunofluorescence staining. Agreement (Cohen's κ) and concordance between the three algorithms were assessed in patients with suspicion of PCD referred to our diagnostic centre. RESULTS: In 46 out of 54 patients (85%) the final diagnosis was concordant between all three algorithms (30 PCD negative, 16 PCD positive). In eight patients (15%) PCD diagnosis differed between the algorithms. Five patients (9%) were diagnosed as PCD only by the ATS, one (2%) only by the ERS and PCD-UNIBE, one (2%) only by the ATS and PCD-UNIBE, and one (2%) only by the PCD-UNIBE algorithm. Agreement was substantial between the ERS and the ATS (κ=0.72, 95% CI 0.53-0.92) and the ATS and the PCD-UNIBE (κ=0.73, 95% CI 0.53-0.92) and almost perfect between the ERS and the PCD-UNIBE algorithms (κ=0.92, 95% CI 0.80-1.00). CONCLUSION: The different diagnostic algorithms lead to a contradictory diagnosis in a considerable proportion of patients. Thus, an updated, internationally harmonised and standardised PCD diagnostic algorithm is needed to improve diagnostics for these discordant cases.

A Comprehensive Approach for the Diagnosis of Primary Ciliary Dyskinesia-Experiences from the First 100 Patients of the PCD-UNIBE Diagnostic Center.

Primary ciliary dyskinesia (PCD) is a rare genetic disease characterized by dyskinetic cilia. Respiratory symptoms usually start at birth. The lack of diagnostic gold standard tests is challenging, as PCD diagnostics requires different methods with high expertise. We founded PCD-UNIBE as the first comprehensive PCD diagnostic center in Switzerland. Our diagnostic approach includes nasal brushing and cell culture with analysis of ciliary motility via high-speed-videomicroscopy (HSVM) and immunofluorescence labeling (IF) of structural proteins. Selected patients undergo electron microscopy (TEM) of ciliary ultrastructure and genetics. We report here on the first 100 patients assessed by PCD-UNIBE. All patients received HSVM fresh, IF, and cell culture (success rate of 90%). We repeated the HSVM with cell cultures and conducted TEM in 30 patients and genetics in 31 patients. Results from cell cultures were much clearer compared to fresh samples. For 80 patients, we found no evidence of PCD, 17 were diagnosed with PCD, two remained inconclusive, and one case is ongoing. HSVM was diagnostic in 12, IF in 14, TEM in five and genetics in 11 cases. None of the methods was able to diagnose all 17 PCD cases, highlighting that a comprehensive approach is essential for an accurate diagnosis of PCD.

Diesel exposure increases susceptibility of primary human nasal epithelial cells to rhinovirus infection.

Nasal epithelial cells (NECs) are among the first cells to be exposed to air pollutants and respiratory viruses. Although it is known that air pollution exposure and rhinovirus infections increase the risk for asthma development independently, it is unclear how these risk factors interact on a cellular level. Therefore, we aimed to investigate how exposure to diesel particulate matter (DPM) modifies the response of primary NECs to rhinovirus (RV) infection in vitro. Exposure of re-differentiated, primary NECs (49 healthy children [0-7 years], 12 adults) to DPM modified the mRNA expression of viral cell-surface receptors, pattern recognition receptors, and pro-inflammatory response (also protein levels). After exposure to DPM, we additionally infected the NECs with RV-1b and RV-16. Viral loads (assessed by titration assays) were significantly higher in DPM-exposed compared with non-exposed NECs. Exposure to DPM prior to RV infection resulted in a significant upregulation of pro-inflammatory cytokines (mRNA and protein level) and ß-defensins mRNA, and significant downregulation of pattern recognition receptors mRNA and CXCL10 (mRNA and protein levels). There was no difference between all outcomes of NECs from children and adults. We can conclude that exposure to DPM prior to RV infection increases viral loads by downregulation of viral defense receptors and upregulation of pro-inflammatory cytokines. Our findings indicate a strong interaction between air pollution and the antiviral response to RV infection in NECs. We provide mechanistic evidence that exposure to air pollution increases susceptibility to RV infection.

Nasal microbiota and symptom persistence in acute respiratory tract infections in infants.

Acute respiratory tract infections (ARI) in infancy have been implicated in the development of chronic respiratory disease, but the complex interplay between viruses, bacteria and host is not completely understood. We aimed to prospectively determine whether nasal microbiota changes occur between the onset of the first symptomatic ARI in the first year of life and 3 weeks later, and to explore possible associations with the duration of respiratory symptoms, as well as with host, environmental and viral factors. Nasal microbiota of 167 infants were determined at both time-points by 16S ribosomal RNA-encoding gene PCR amplification and subsequent pyrosequencing. Infants were clustered based on their nasal microbiota using hierarchical clustering methods at both time-points. We identified five dominant infant clusters with distinct microbiota at the onset of ARI but only three clusters after 3 weeks. In these three clusters, symptom persistence was overrepresented in the Streptococcaceae-dominated cluster and underrepresented in the cluster dominated by "Others" (p<0.001). Duration of symptoms was not associated with the type of respiratory virus. Infants with prolonged respiratory symptoms after their first ARI tend to exhibit distinct microbial compositions, indicating close microbiota-host interactions that seem to be of importance for symptom persistence and recovery.

Response of cord blood cells to environmental, hereditary and perinatal factors: A prospective birth cohort study.

BACKGROUND: Many studies investigating the impact of individual risk factors on cord blood immune cell counts may be biased given that cord blood composition is influenced by a multitude of factors. The aim of this study was to comprehensively investigate the relative impact of environmental, hereditary and perinatal factors on cord blood cells. METHODS: In 295 neonates from the prospective Basel-Bern Infant Lung Development Cohort, we performed complete blood counts and fluorescence-activated cell sorting scans of umbilical cord blood. The associations between risk factors and cord blood cells were assessed using multivariable linear regressions. RESULTS: The multivariable regression analysis showed that an increase per 10µg/m3 of the average nitrogen dioxide 14 days before birth was associated with a decrease in leukocyte (6.7%, 95% CI:-12.1,-1.0) and monocyte counts (11.6%, 95% CI:-19.6,-2.8). Maternal smoking during pregnancy was associated with significantly lower cord blood cell counts in multiple cell populations. Moreover, we observed sex differences regarding eosinophilic granulocytes and plasmacytoid dendritic cells. Finally, significantly increased numbers of cord blood cells were observed in infants exposed to perinatal stress. Cesarean section seems to play a significant role in Th1/Th2 balance. CONCLUSIONS: Our results suggest that all three: environmental, hereditary and perinatal factors play a significant role in the composition of cord blood cells at birth, and it is important to adjust for all of these factors in cord blood studies. In particular, perinatal circumstances seem to influence immune balance, which could have far reaching consequences in the development of immune mediated diseases.

Gasoline particle filter reduces oxidative DNA damage in bronchial epithelial cells after whole gasoline exhaust exposure in vitro.

A substantial amount of traffic-related particle emissions is released by gasoline cars, since most diesel cars are now equipped with particle filters that reduce particle emissions. Little is known about adverse health effects of gasoline particles, and particularly, whether a gasoline particle filter (GPF) influences the toxicity of gasoline exhaust emissions. We drove a dynamic test cycle with a gasoline car and studied the effect of a GPF on exhaust composition and airway toxicity. We exposed human bronchial epithelial cells (ECs) for 6 hours, and compared results with and without GPF. Two hours later, primary human natural killer cells (NKs) were added to ECs to form cocultures, while some ECs were grown as monocultures. The following day, cells were analyzed for cytotoxicity, cell surface receptor expression, intracellular markers, oxidative DNA damage, gene expression, and oxidative stress. The particle amount was significantly reduced due to GPF application. While most biological endpoints did not differ, oxidative DNA damage was significantly reduced in EC monocultures exposed to GPF compared to reference exhaust. Our findings indicate that a GPF has beneficial effects on exhaust composition and airway toxicity. Further studies are needed to assess long-term effects, also in other cell types of the lung.

Assessment of lung cell toxicity of various gasoline engine exhausts using a versatile in vitro exposure system.

Adverse effect studies of gasoline exhaust are scarce, even though gasoline direct injection (GDI) vehicles can emit a high number of particles. The aim of this study was to conduct an in vitro hazard assessment of different GDI exhausts using two different cell culture models mimicking the human airway. In addition to gasoline particle filters (GPF), the effects of two lubrication oils with low and high ash content were assessed, since it is known that oils are important contributors to exhaust emissions. Complete exhausts from two gasoline driven cars (GDI1 and GDI2) were applied for 6 h (acute exposure) to a multi-cellular human lung model (16HBE14o-cell line, macrophages, and dendritic cells) and a primary human airway model (MucilAir™). GDI1 vehicle was driven unfiltered and filtered with an uncoated and a coated GPF. GDI2 vehicle was driven under four settings with different fuels: normal unleaded gasoline, 2% high and low ash oil in gasoline, and 2% high ash oil in gasoline with a GPF. GDI1 unfiltered was also used for a repeated exposure (3 times 6 h) to assess possible adverse effects. After 6 h exposure, no genes or proteins for oxidative stress or pro-inflammation were upregulated compared to the filtered air control in both cell systems, neither in GDI1 with GPFs nor in GDI2 with the different fuels. However, the repeated exposure led to a significant increase in HMOX1 and TNFa gene expression in the multi-cellular model, showing the responsiveness of the system towards gasoline engine exhaust upon prolonged exposure. The reduction of particles by GPFs is significant and no adverse effects were observed in vitro during a short-term exposure. On the other hand, more data comparing different lubrication oils and their possible adverse effects are needed. Future experiments also should, as shown here, focus on repeated exposures.

Exposure to silver nanoparticles affects viability and function of natural killer cells, mostly via the release of ions.

Natural killer (NK) cells play a crucial role in linking innate and adaptive immune responses, especially during viral infections and tumor surveillance. They have two major effector functions: the killing of stressed/abnormal cells and the release of cytokines. Their activity is regulated via inhibitory and activating surface receptors. At the same time that the production and use of engineered nanoparticles is steadily increasing, the risk for exposure to silver nanoparticles (AgNPs) from consumer products or biomedical applications is growing. Given this, we assessed the effects of 20-nm big AgNPs on NK cells, which represent an important part of the immune system. Our study involved overnight exposure of human blood NK cells to different concentrations of AgNPs, and silver (Ag) ion controls, and analyzing them for viability, surface receptor expression, intracellular markers, cytokine release, and killing potential. Exposure to AgNPs, but not to Ag ion controls, reduced the viability and the cytotoxic potential after polyriboinosinic-polyribocytidylic acid stimulation of NK cells and increased the expression of the inhibitory receptor CD159a. Exposure to AgNPs and Ag ion controls reduced the expression of the activating receptors CD335 and of CD16 and increased the expression of the activating receptor CD314. Overall, exposure to AgNPs changes NK cells' function and phenotype and may present a risk for modulating human immune responses, which should be further investigated.

Effects of gasoline and ethanol-gasoline exhaust exposure on human bronchial epithelial and natural killer cells in vitro.

Air pollution exposure, including passenger car emissions, may cause substantial respiratory health effects and cancer death. In western countries, the majority of passenger cars are driven by gasoline fuel. Recently, new motor technologies and ethanol fuels have been introduced to the market, but potential health effects have not been thoroughly investigated. We developed and verified a coculture model composed of bronchial epithelial cells (ECs) and natural killer cells (NKs) mimicking the human airways to compare toxic effects between pure gasoline (E0) and ethanol-gasoline-blend (E85, 85% ethanol, 15% gasoline) exhaust emitted from a flexfuel gasoline car. We drove a steady state cycle, exposed ECs for 6h and added NKs. We assessed exhaust effects in ECs alone and in cocultures by RT-PCR, flow cytometry, and oxidative stress assay. We found no toxic effects after exposure to E0 or E85 compared to air controls. Comparison between E0 and E85 exposure showed a weak association for less oxidative DNA damage after E85 exposure compared to E0. Our results indicate that short-term exposure to gasoline exhaust may have no major toxic effects in ECs and NKs and that ethanol as part of fuel for gasoline cars may be favorable.

Hazard identification of exhausts from gasoline-ethanol fuel blends using a multi-cellular human lung model.

Ethanol can be produced from biomass and as such is renewable, unlike petroleum-based fuel. Almost all gasoline cars can drive with fuel containing 10% ethanol (E10), flex-fuel cars can even use 85% ethanol (E85). Brazil and the USA already include 10-27% ethanol in their standard fuel by law. Most health effect studies on car emissions are however performed with diesel exhausts, and only few data exists for other fuels. In this work we investigated possible toxic effects of exhaust aerosols from ethanol-gasoline blends using a multi-cellular model of the human lung. A flex-fuel passenger car was driven on a chassis dynamometer and fueled with E10, E85, or pure gasoline (E0). Exhausts obtained from a steady state cycle were directly applied for 6h at a dilution of 1:10 onto a multi-cellular human lung model mimicking the bronchial compartment composed of human bronchial cells (16HBE14o-), supplemented with human monocyte-derived dendritic cells and monocyte-derived macrophages, cultured at the air-liquid interface. Biological endpoints were assessed after 6h post incubation and included cytotoxicity, pro-inflammation, oxidative stress, and DNA damage. Filtered air was applied to control cells in parallel to the different exhausts; for comparison an exposure to diesel exhaust was also included in the study. No differences were measured for the volatile compounds, i.e. CO, NOx, and T.HC for the different ethanol supplemented exhausts. Average particle number were 6×102 #/cm3 (E0), 1×105 #/cm3 (E10), 3×103 #/cm3 (E85), and 2.8×106 #/cm3 (diesel). In ethanol-gasoline exposure conditions no cytotoxicity and no morphological changes were observed in the lung cell cultures, in addition no oxidative stress - as analyzed with the glutathione assay - was measured. Gene expression analysis also shows no induction in any of the tested genes, including mRNA levels of genes related to oxidative stress and pro-inflammation, as well as indoleamine 2,3-dioxygenase 1 (IDO-1), transcription factor NFE2-related factor 2 (NFE2L2), and NAD(P)H dehydrogenase [quinone] 1 (NQO1). Finally, no DNA damage was observed with the OxyDNA assay. On the other hand, cell death, oxidative stress, as well as an increase in pro-inflammatory cytokines was observed for cells exposed to diesel exhaust, confirming the results of other studies and the applicability of our exposure system. In conclusion, the tested exhausts from a flex-fuel gasoline vehicle using different ethanol-gasoline blends did not induce adverse cell responses in this acute exposure. So far ethanol-gasoline blends can promptly be used, though further studies, e.g. chronic and in vivo studies, are needed.