High-quality identification of volatile organic compounds (VOCs) originating from breath.

INTRODUCTION: Volatile organic compounds (VOCs) can arise from underlying metabolism and are detectable in exhaled breath, therefore offer a promising route to non-invasive diagnostics. Robust, precise, and repeatable breath measurement platforms able to identify VOCs in breath distinguishable from background contaminants are needed for the confident discovery of breath-based biomarkers. OBJECTIVES: To build a reliable breath collection and analysis method that can produce a comprehensive list of known VOCs in the breath of a heterogeneous human population. METHODS: The analysis cohort consisted of 90 pairs of breath and background samples collected from a heterogenous population. Owlstone Medical's Breath Biopsy® OMNI® platform, consisting of sample collection, TD-GC-MS analysis and feature extraction was utilized. VOCs were determined to be "on-breath" if they met at least one of three pre-defined metrics compared to paired background samples. On-breath VOCs were identified via comparison against purified chemical standards, using retention indexing and high-resolution accurate mass spectral matching. RESULTS: 1471 VOCs were present in > 80% of samples (breath and background), and 585 were on-breath by at least one metric. Of these, 148 have been identified covering a broad range of chemical classes. CONCLUSIONS: A robust breath collection and relative-quantitative analysis method has been developed, producing a list of 148 on-breath VOCs, identified using purified chemical standards in a heterogenous population. Providing confirmed VOC identities that are genuinely breath-borne will facilitate future biomarker discovery and subsequent biomarker validation in clinical studies. Additionally, this list of VOCs can be used to facilitate cross-study data comparisons for improved standardization.

Assay conditions for estimating differences in base excision repair activity with Fpg-modified comet assay.

DNA repair is an essential agent in cancer development, progression, prognosis, and response to therapy. We have adapted a cellular repair assay based on the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay to assess DNA repair kinetics. The removal of oxidized nucleobases over time (0-480 min) was analyzed in peripheral blood mononuclear cells (PBMCs) and 8 cell lines. DNA damage was induced by exposure to either Ro19-8022 plus visible light or potassium bromate (KBrO3). The initial amount of damage induced by Ro 19-8022 plus light varied between cell lines, and this was apparently associated with the rate of repair. However, the amount of DNA damage induced by KBrO3 varied less between cell types, so we used this agent to study the kinetics of DNA repair. We found an early phase of ca. 60 min with fast removal of Fpg-sensitive sites, followed by slower removal over the following 7 h. In conclusion, adjusting the initial damage at T0 to an equal level can be achieved by the use of KBrO3, which allows for accurate analysis of subsequent cellular DNA repair kinetics in the first hour after exposure.

Comparison of comet-based approaches to assess base excision repair.

DNA repair plays an essential role in maintaining genomic stability, and can be assessed by various comet assay-based approaches, including the cellular repair assay and the in vitro repair assay. In the cellular repair assay, cells are challenged with a DNA-damaging compound and DNA damage removal over time is assessed. In the in vitro repair assay, an early step in the repair process is assessed as the ability of a cellular extract to recognize and incise damaged DNA in substrate nucleoids from cells treated with a DNA-damaging compound. Our direct comparison of both assays in eight cell lines and human peripheral blood lymphocytes indicated no significant relationship between these DNA repair assays (R2 = 0.084, P = 0.52). The DNA incision activity of test cells measured with the in vitro repair assay correlated with the background level of DNA damage in the untreated test cells (R2 = 0.621, P = 0.012). When extracts were prepared from cells exposed to DNA-damaging agents (10 mM KBrO3 or 1 µM Ro 19-8022 plus light), the incision activity was significantly increased, which is in line with the notion that base excision repair is inducible. The data presented suggest that the two assays do not measure the same endpoint of DNA repair and should be considered as complementary.

The ADEM2 project: early pathogenic mechanisms of preschool wheeze and a randomised controlled trial assessing the gain in health and cost-effectiveness by application of the breath test for the diagnosis of asthma in wheezing preschool children.

BACKGROUND: The prevalence of asthma-like symptoms in preschool children is high. Despite numerous efforts, there still is no clinically available diagnostic tool to discriminate asthmatic children from children with transient wheeze at preschool age. This leads to potential overtreatment of children outgrowing their symptoms, and to potential undertreatment of children who turn out to have asthma. Our research group developed a breath test (using GC-tof-MS for VOC-analysis in exhaled breath) that is able to predict a diagnosis of asthma at preschool age. The ADEM2 study assesses the improvement in health gain and costs of care with the application of this breath test in wheezing preschool children. METHODS: This study is a combination of a multi-centre, parallel group, two arm, randomised controlled trial and a multi-centre longitudinal observational cohort study. The preschool children randomised into the treatment arm of the RCT receive a probability diagnosis (and corresponding treatment recommendations) of either asthma or transient wheeze based on the exhaled breath test. Children in the usual care arm do not receive a probability diagnosis. Participants are longitudinally followed up until the age of 6 years. The primary outcome is disease control after 1 and 2 years of follow-up. Participants of the RCT, together with a group of healthy preschool children, also contribute to the parallel observational cohort study developed to assess the validity of alternative VOC-sensing techniques and to explore numerous other potential discriminating biological parameters (such as allergic sensitisation, immunological markers, epigenetics, transcriptomics, microbiomics) and the subsequent identification of underlying disease pathways and relation to the discriminative VOCs in exhaled breath. DISCUSSION: The potential societal and clinical impact of the diagnostic tool for wheezing preschool children is substantial. By means of the breath test, it will become possible to deliver customized and high qualitative care to the large group of vulnerable preschool children with asthma-like symptoms. By applying a multi-omics approach to an extensive set of biological parameters we aim to explore (new) pathogenic mechanisms in the early development of asthma, creating potentially interesting targets for the development of new therapies. TRIAL REGISTRATION: Netherlands Trial Register, NL7336, Date registered 11-10-2018.

Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients.

The comet assay-based in vitro DNA repair assay has become a common tool for quantifying base excision repair (BER) activity in human lymphocytes or cultured cells. Here, we optimized the protocol for studying BER in human placental tissue because the placenta is a non-invasive tissue for biomonitoring of early-life exposures, and it can be used to investigate molecular mechanisms associated with prenatal disorders. The optimal protein concentration of placental protein extracts for optimal damage recognition and incision was 2 mg protein/mL. The addition of aphidicolin did not lead to reduced non-specific incisions and was, therefore, not included in the optimized protocol. The interval between sample collection and analysis did not affect BER activity up to 70 min. Finally, this optimized protocol was tested on pre-eclamptic (PE) placental tissues (n = 11) and significantly lower BER activity in PE placentas compared to controls (n = 9) was observed. This was paralleled by a significant reduction in the expression of BER-related genes and increased DNA oxidation in PE placentas. Our study indicates that BER activity can be determined in placentas, and lower activity is present in PE compared with healthy. These findings should be followed up in prospective clinical investigations to examine BER's role in the advancement of PE.

Upregulation of mNEIL3 in Ogg1-null cells is a potential backup mechanism for 8-oxoG repair.

Reactive oxygen species formation and resultant oxidative damage to DNA are ubiquitous events in cells, the homeostasis of which can be dysregulated in a range of pathological conditions. Base excision repair (BER) is the primary repair mechanism for oxidative genomic DNA damage. One prevalent oxidised base modification, 8-oxoguanine (8-oxoG), is recognised by 8-oxoguanine glycosylase-1 (OGG1) initiating removal and repair via BER. Surprisingly, Ogg1 null mouse embryonic fibroblasts (mOgg1-/- MEFs) do not accumulate 8-oxoG in the genome to the extent expected. This suggests that there are backup repair mechanisms capable of repairing 8-oxoG in the absence of OGG1. In the current study, we identified components of NER (Ercc1, Ercc4, Ercc5), BER (Lig1, Tdg, Nthl1, Mpg, Mgmt, NEIL3), MMR (Mlh1, Msh2, Msh6) and DSB (Brip1, Rad51d, Prkdc) pathways that are transcriptionally elevated in mOgg1-/- MEFs. Interestingly, all three nucleotide excision repair genes identified: Ercc1 (2.5 ± 0.2-fold), Ercc4 (1.5 ± 0.1-fold) and Ercc5 (1.7 ± 0.2-fold) have incision activity. There was also a significant functional increase in NER activity (42.0 ± 7.9%) compared to WT MEFs. We also observed upregulation of both Neil3 mRNA (37.9 ± 1.6-fold) and protein in mOgg1-/- MEFs. This was associated with a 3.4 ± 0.4-fold increase in NEIL3 substrate sites in genomic DNA of cells treated with BSO, consistent with the ability of NEIL3 to remove 8-oxoG oxidation products from genomic DNA. In conclusion, we suggest that in Ogg1-null cells, upregulation of multiple DNA repair proteins including incision components of the NER pathway and Neil3 are important compensatory responses to prevent the accumulation of genomic 8-oxoG.

Exhaled Volatile Organic Compounds Are Able to Discriminate between Neutrophilic and Eosinophilic Asthma.

Rationale: Analysis of exhaled breath for asthma phenotyping using endogenously generated volatile organic compounds (VOCs) offers the possibility of noninvasive diagnosis and therapeutic monitoring. Induced sputum is indeed not widely available and markers of neutrophilic asthma are still lacking.Objectives: To determine whether analysis of exhaled breath using endogenously generated VOCs can be a surrogate marker for recognition of sputum inflammatory phenotypes.Methods: We conducted a prospective study on 521 patients with asthma recruited from the University Asthma Clinic of Liege. Patients underwent VOC measurement, fraction of exhaled nitric oxide (FeNO) spirometry, sputum induction, and gave a blood sample. Subjects with asthma were classified in three inflammatory phenotypes according to their sputum granulocytic cell count.Measurements and Main Results: In the discovery study, seven potential biomarkers were highlighted by gas chromatography-mass spectrometry in a training cohort of 276 patients with asthma. In the replication study (n = 245), we confirmed four VOCs of interest to discriminate among asthma inflammatory phenotypes using comprehensive two-dimensional gas chromatography coupled to high-resolution time-of-flight mass spectrometry. Hexane and 2-hexanone were identified as compounds with the highest classification performance in eosinophilic asthma with accuracy comparable to that of blood eosinophils and FeNO. Moreover, the combination of FeNO, blood eosinophils, and VOCs gave a very good prediction of eosinophilic asthma (area under the receiver operating characteristic curve, 0.9). For neutrophilic asthma, the combination of nonanal, 1-propanol, and hexane had a classification performance similar to FeNO or blood eosinophils in eosinophilic asthma. Those compounds were found in higher levels in neutrophilic asthma.Conclusions: Our study is the first attempt to characterize VOCs according to sputum granulocytic profile in a large population of patients with asthma and provide surrogate markers for neutrophilic asthma.

The dietary antioxidant quercetin reduces hallmarks of bleomycin-induced lung fibrogenesis in mice.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, lethal disease of which the etiology is still not fully understood. Current treatment comprises two FDA-approved drugs that can slow down yet not stop or reverse the disease. As IPF pathology is associated with an altered redox balance, adding a redox modulating component to current therapy might exert beneficial effects. Quercetin is a dietary antioxidant with strong redox modulating capacities that is suggested to exert part of its antioxidative effects via activation of the redox-sensitive transcription factor Nrf2 that regulates endogenous antioxidant levels. Therefore, the aim of the present study was to investigate if the dietary antioxidant quercetin can exert anti-fibrotic effects in a mouse model of bleomycin-induced pulmonary fibrogenesis through Nrf2-dependent restoration of redox imbalance. METHODS: Homozygous Nrf2 deficient mice and their wildtype littermates were fed a control diet without or with 800 mg quercetin per kg diet from 7 days prior to a single 1 µg/2 µl per g BW bleomycin challenge until they were sacrificed 14 days afterwards. Lung tissue and plasma were collected to determine markers of fibrosis (expression of extracellular matrix genes and histopathology), inflammation (pulmonary gene expression and plasma levels of tumor necrosis factor-α (TNFα) and keratinocyte chemoattrachtant (KC)), and redox balance (pulmonary gene expression of antioxidants and malondialdehyde-dG (MDA)- DNA adducts). RESULTS: Mice fed the enriched diet for 7 days prior to the bleomycin challenge had significantly enhanced plasma and pulmonary quercetin levels (11.08 ± 0.73 µM versus 7.05 ± 0.2 µM) combined with increased expression of Nrf2 and Nrf2-responsive genes compared to mice fed the control diet in lung tissue. Upon bleomycin treatment, quercetin-fed mice displayed reduced expression of collagen (COL1A2) and fibronectin (FN1) and a tendency of reduced inflammatory lesions (2.8 ± 0.7 versus 1.9 ± 0.8). These beneficial effects were accompanied by reduced pulmonary gene expression of TNFα and KC, but not their plasma levels, and enhanced Nrf2-induced pulmonary antioxidant defences. In Nrf2 deficient mice, no effect of the dietary antioxidant on either histology or inflammatory lesions was observed. CONCLUSION: Quercetin exerts anti-fibrogenic and anti-inflammatory effects on bleomycin-induced pulmonary damage in mice possibly through modulation of the redox balance by inducing Nrf2. However, quercetin could not rescue the bleomycin-induced pulmonary damage indicating that quercetin alone cannot ameliorate the progression of IPF.

Ligation-Mediated Polymerase Chain Reaction Detection of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine and 5-Hydroxycytosine at the Codon 176 of the p53 Gene of Hepatitis C-Associated Hepatocellular Carcinoma Patients.

Molecular mechanisms underlying Hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) pathogenesis are still unclear. Therefore, we analyzed the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and other oxidative lesions at codon 176 of the p53 gene, as well as the generation of 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG), in a cohort of HCV-related HCC patients from Italy. Detection of 8-oxodG and 5-hydroxycytosine (5-OHC) was performed by ligation mediated-polymerase chain reaction assay, whereas the levels of M1dG were measured by chromatography and mass-spectrometry. Results indicated a significant 130% excess of 8-oxodG at -TGC- position of p53 codon 176 in HCV-HCC cases as compared to controls, after correction for age and gender, whereas a not significant increment of 5-OHC at -TGC- position was found. Then, regression models showed an 87% significant excess of M1dG in HCV-HCC cases relative to controls. Our study provides evidence that increased adduct binding does not occur randomly on the sequence of the p53 gene but at specific sequence context in HCV-HCC patients. By-products of lipid peroxidation could also yield a role in HCV-HCC development. Results emphasize the importance of active oxygen species in inducing nucleotide lesions at a p53 mutational hotspot in HCV-HCC patients living in geographical areas without dietary exposure to aflatoxin B1.

Interaction between dietary acrylamide intake and genetic variants for estrogen receptor-positive breast cancer risk.

PURPOSE: The association between dietary acrylamide intake and estrogen receptor-positive (ER+) breast cancer risk in epidemiological studies is inconsistent. By analyzing gene-acrylamide interactions for ER+ breast cancer risk, we aimed to clarify the role of acrylamide intake in ER+ breast cancer etiology. METHODS: The prospective Netherlands Cohort Study on diet and cancer includes 62,573 women, aged 55-69 years. At baseline, a random subcohort of 2589 women was sampled from the total cohort for a case-cohort analysis approach. Dietary acrylamide intake of subcohort members (n = 1449) and ER+ breast cancer cases (n = 844) was assessed with a food frequency questionnaire. We genotyped single nucleotide polymorphisms (SNPs) in genes in acrylamide metabolism, sex steroid systems, oxidative stress and DNA repair. Multiplicative interaction between acrylamide intake and SNPs was assessed with Cox proportional hazards analysis, based on 20.3 years of follow-up. RESULTS: Unexpectedly, there was a statistically non-significant inverse association between acrylamide and ER+ breast cancer risk among all women but with no clear dose-response relationship, and no association among never smokers. Among the results for 57 SNPs and 2 gene deletions, rs1056827 in CYP1B1, rs2959008 and rs7173655 in CYP11A1, the GSTT1 gene deletion, and rs1052133 in hOGG1 showed a statistically significant interaction with acrylamide intake for ER+ breast cancer risk. CONCLUSIONS: This study did not provide evidence for a positive association between acrylamide intake and ER+ breast cancer risk. If anything, acrylamide was associated with a decreased ER+ breast cancer risk. The interaction with SNPs in CYP1B1 and CYP11A1 suggests that acrylamide may influence ER+ breast cancer risk through sex hormone pathways.

Fruit and vegetable intake and the risk of recurrence in patients with non-muscle invasive bladder cancer: a prospective cohort study.

INTRODUCTION: There is some evidence that greater consumption of fruit and vegetables decreases the risk of bladder cancer. The role of fruit and vegetables in bladder cancer recurrence is still unknown. OBJECTIVE: The role of total fruit and vegetable intake in relation to the risk of developing bladder cancer recurrence in a prospective cohort study. METHODS: 728 patients with non-muscle invasive bladder cancer (NMIBC), who completed self-administrated questionnaires on fruit and vegetable intake at time of diagnosis (over the year before diagnosis) and 1 year after diagnosis, were included. Hazard ratios and 95% confidence intervals were calculated by multivariable Cox regression for developing recurrent bladder cancer in relation to fruit and vegetable intake. RESULTS: During 2,051 person-years of follow-up [mean (SD) follow-up 3.7 (1.5) years], 241 (33.1%) of the included 728 NMIBC patients developed a recurrence of bladder cancer. The sum of total fruit and vegetables before diagnosis was not related to a first bladder cancer recurrence (HR 1.07; 95% CI 0.78-1.47, p = 0.66). No association was found between greater consumption of fruit and vegetables over the year before diagnosis and the risk of developing multiple recurrences of bladder cancer (HR 1.02; 95% CI 0.90-1.15, p = 0.78). Among the remaining 389 NMIBC patients who reported on fruit and vegetable intake 1 year after diagnosis, no association was found between greater consumption of fruit and vegetables and a first recurrence of bladder cancer (HR 0.65; 95% CI 0.42-1.01, p = 0.06) nor with multiple recurrences of bladder cancer (HR 1.00, 95% CI 0.85-1.18, p = 1.00). Similar results were obtained when investigating the association between total intakes of fruit and vegetables separately and bladder cancer recurrence. CONCLUSION: Results from this study did not indicate a protective role for total fruit and vegetables in the development of a recurrence of NMIBC.

The Role of Genetic Variants in the Association between Dietary Acrylamide and Advanced Prostate Cancer in the Netherlands Cohort Study on Diet and Cancer.

To investigate the association between dietary acrylanide and advanced prostate cancer, we examined acrylamide-gene interactions for advanced prostate cancer risk by using data from the Netherlands Cohort Study. Participants (n = 58,279 men) completed a baseline food frequency questionnaire (FFQ), from which daily acrylamide intake was calculated. At baseline, 2,411 men were randomly selected from the full cohort for case-cohort analysis. Fifty eight selected single nucleotide polymorphisms (SNPs) and two gene deletions in genes in acrylamide metabolism, DNA repair, sex steroid systems, and oxidative stress were analyzed. After 20.3 years of follow-up, 1,608 male subcohort members and 948 advanced prostate cancer cases were available for Cox analysis. Three SNPs showed a main association with advanced prostate cancer risk after multiple testing correction: catalase (CAT) rs511895, prostaglandin-endoperoxide synthase 2 (PTGS2) rs5275, and xeroderma pigmentosum group C (XPC) rs2228001. With respect to acrylamide-gene interactions, only rs1800566 in NAD(P)H quinone dehydrogenase 1 (NQO1) and rs2301241 in thioredoxin (TXN) showed a nominally statistically significant multiplicative interaction with acrylamide intake for advanced prostate cancer risk. After multiple testing corrections, none were statistically significant. In conclusion, no clear evidence was found for interaction between acrylamide intake and selected genetic variants for advanced prostate cancer risk.

A European Respiratory Society technical standard: exhaled biomarkers in lung disease.

Breath tests cover the fraction of nitric oxide in expired gas (FeNO), volatile organic compounds (VOCs), variables in exhaled breath condensate (EBC) and other measurements. For EBC and for FeNO, official recommendations for standardised procedures are more than 10 years old and there is none for exhaled VOCs and particles. The aim of this document is to provide technical standards and recommendations for sample collection and analytic approaches and to highlight future research priorities in the field. For EBC and FeNO, new developments and advances in technology have been evaluated in the current document. This report is not intended to provide clinical guidance on disease diagnosis and management.Clinicians and researchers with expertise in exhaled biomarkers were invited to participate. Published studies regarding methodology of breath tests were selected, discussed and evaluated in a consensus-based manner by the Task Force members.Recommendations for standardisation of sampling, analysing and reporting of data and suggestions for research to cover gaps in the evidence have been created and summarised.Application of breath biomarker measurement in a standardised manner will provide comparable results, thereby facilitating the potential use of these biomarkers in clinical practice.

Interactions between dietary acrylamide intake and genes for ovarian cancer risk.

Some epidemiological studies observed a positive association between dietary acrylamide intake and ovarian cancer risk but the causality needs to be substantiated. By analyzing gene-acrylamide interactions for ovarian cancer risk for the first time, we aimed to contribute to this. The prospective Netherlands Cohort Study on diet and cancer includes 62,573 women, aged 55-69 years. At baseline in 1986, a random subcohort of 2589 women was sampled from the total cohort for a case cohort analysis approach. Dietary acrylamide intake of subcohort members and ovarian cancer cases (n = 252, based on 20.3 years of follow-up) was assessed with a food frequency questionnaire. We selected single nucleotide polymorphisms (SNPs) in genes in acrylamide metabolism and in genes involved in the possible mechanisms of acrylamide-induced carcinogenesis (effects on sex steroid systems, oxidative stress and DNA damage). Genotyping was done on DNA from toenails through Agena's MassARRAY iPLEX platform. Multiplicative interaction between acrylamide intake and SNPs was assessed with Cox proportional hazards analysis. Among the results for 57 SNPs and 2 gene deletions, there were no statistically significant interactions between acrylamide and gene variants after adjustment for multiple testing. However, there were several nominally statistically significant interactions between acrylamide intake and SNPs in the HSD3B1/B2 gene cluster: (rs4659175 (p interaction = 0.04), rs10923823 (p interaction = 0.06) and its proxy rs7546652 (p interaction = 0.05), rs1047303 (p interaction = 0.005), and rs6428830 (p interaction = 0.05). Although in need of confirmation, results of this study suggest that acrylamide may cause ovarian cancer through effects on sex hormones.

Polymorphisms in genes of the renin-angiotensin-aldosterone system and renal cell cancer risk: interplay with hypertension and intakes of sodium, potassium and fluid.

Hypertension is an established risk factor for renal cell cancer (RCC). The renin-angiotensin-aldosterone system (RAAS) regulates blood pressure and is closely linked to hypertension. RAAS additionally influences homeostasis of electrolytes (e.g. sodium and potassium) and fluid. We investigated single nucleotide polymorphisms (SNPs) in RAAS and their interactions with hypertension and intakes of sodium, potassium and fluid regarding RCC risk in the Netherlands Cohort Study (NLCS), which was initiated in 1986 and included 120,852 participants aged 55 to 69 years. Diet and lifestyle were assessed by questionnaires and toenail clippings were collected. Genotyping of toenail DNA was performed using the SEQUENOM® MassARRAY® platform for a literature-based selection of 13 candidate SNPs in seven key RAAS genes. After 20.3 years of follow-up, Cox regression analyses were conducted using a case-cohort approach including 3,583 subcohort members and 503 RCC cases. Two SNPs in AGTR1 were associated with RCC risk. AGTR1_rs1492078 (AA vs. GG) decreased RCC risk [hazard ratio (HR) (95% confidence interval (CI)): 0.70(0.49-1.00)], whereas AGTR1_rs5186 (CC vs. AA) increased RCC risk [HR(95%CI): 1.49(1.08-2.05)]. Associations were stronger in participants with hypertension. The RCC risk for AGT_rs3889728 (AG + AA vs. GG) was modified by hypertension (p interaction = 0.039). SNP-diet interactions were not significant, although HRs suggested interaction between SNPs in ACE and sodium intake. SNPs in AGTR1 and AGT influenced RCC susceptibility, and their effects were modified by hypertension. Sodium intake was differentially associated with RCC risk across genotypes of several SNPs, yet some analyses had probably inadequate power to show significant interaction. Results suggest that RAAS may be a candidate pathway in RCC etiology.

Defining adult asthma endotypes by clinical features and patterns of volatile organic compounds in exhaled air.

BACKGROUND: Several classifications of adult asthma patients using cluster analyses based on clinical and demographic information has resulted in clinical phenotypic clusters that do not address molecular mechanisms. Volatile organic compounds (VOC) in exhaled air are released during inflammation in response to oxidative stress as a result of activated leukocytes. VOC profiles in exhaled air could distinguish between asthma patients and healthy subjects. In this study, we aimed to classify new asthma endotypes by combining inflammatory mechanisms investigated by VOC profiles in exhaled air and clinical information of asthma patients. METHODS: Breath samples were analyzed for VOC profiles by gas chromatography-mass spectrometry from asthma patients (n = 195) and healthy controls (n = 40). A total of 945 determined compounds were subjected to discriminant analysis to find those that could discriminate healthy from asthmatic subjects. 2-step cluster analysis based on clinical information and VOCs in exhaled air were used to form asthma endotypes. RESULTS: We identified 16 VOCs, which could distinguish between healthy and asthma subjects with a sensitivity of 100% and a specificity of 91.1%. Cluster analysis based on VOCs in exhaled air and the clinical parameters FEV1, FEV1 change after 3 weeks of hospitalization, allergic sensitization, Junipers symptoms score and asthma medications resulted in the formation of 7 different asthma endotype clusters. We identified asthma clusters with different VOC profiles but similar clinical characteristics and endotypes with similar VOC profiles, but distinct clinical characteristics. CONCLUSION: This study demonstrates that both, clinical presentation of asthma and inflammatory mechanisms in the airways should be considered for classification of asthma subtypes.

Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories.

This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).

Integrative genomic analysis identifies a role for intercellular adhesion molecule 1 in childhood asthma.

BACKGROUND: Childhood asthma is characterized by chronic airway inflammation. Integrative genomic analysis of airway inflammation on genetic and protein level may help to unravel mechanisms of childhood asthma. We aimed to employ an integrative genomic approach investigating inflammation markers on DNA, mRNA, and protein level at preschool age in relationship to asthma development. METHODS: In a prospective study, 252 preschool children (202 recurrent wheezers, 50 controls) from the Asthma DEtection and Monitoring (ADEM) study were followed until the age of six. Genetic variants, mRNA expression in peripheral blood mononuclear cells, and protein levels in exhaled breath condensate for intercellular adhesion molecule 1 (ICAM1), interleukin (IL)4, IL8, IL10, IL13, and tumor necrosis factor α were analyzed at preschool age. At six years of age, a classification (healthy, transient wheeze, or asthma) was based on symptoms, lung function, and medication use. RESULTS: The ICAM1 rs5498 A allele was positively associated with asthma development (p = 0.02) and ICAM1 gene expression (p = 0.01). ICAM1 gene expression was positively associated with exhaled levels of soluble ICAM1 (p = 0.04) which in turn was positively associated with asthma development (p = 0.01). Furthermore, rs1800872 and rs1800896 in IL10 were associated with altered IL10 mRNA expression (p < 0.01). Exhaled levels of IL4, IL10, and IL13 were positively associated with asthma development (p < 0.01). CONCLUSIONS: In this unique prospective study, we demonstrated that ICAM1 is associated with asthma development on DNA, mRNA, and protein level. Thus, ICAM1 is likely to be involved in the development of childhood asthma.

The optimization and comparison of two high-throughput faecal headspace sampling platforms: the microchamber/thermal extractor and hi-capacity sorptive extraction probes (HiSorb).

Disease detection and monitoring using volatile organic compounds (VOCs) is becoming increasingly popular. For a variety of (gastrointestinal) diseases the microbiome should be considered. As its output is to large extent volatile, faecal volatilomics carries great potential. One technical limitation is that current faecal headspace analysis requires specialized instrumentation which is costly and typically does not work in harmony with thermal desorption units often utilized in e.g. exhaled breath studies. This lack of harmonization hinders uptake of such analyses by the Volatilomics community. Therefore, this study optimized and compared two recently harmonized faecal headspace sampling platforms:High-capacity Sorptive extraction (HiSorb) probesand theMicrochamber thermal extractor (Microchamber). Statistical design of experiment was applied to find optimal sampling conditions by maximizing reproducibility, the number of VOCs detected, and between subject variation. To foster general applicability those factors were defined using semi-targeted as well as untargeted metabolic profiles. HiSorb probes were found to result in a faster sampling procedure, higher number of detected VOCs, and higher stability. The headspace collection using the Microchamber resulted in a lower number of detected VOCs, longer sampling times and decreased stability despite a smaller number of interfering VOCs and no background signals. Based on the observed profiles, recommendations are provided on pre-processing and study design when using either one of both platforms. Both can be used to perform faecal headspace collection, but altogether HiSorb is recommended.

The potential of volatile organic compounds to diagnose primary sclerosing cholangitis.

Background & Aims: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts. PSC is a complex disease of largely unknown aetiology that is strongly associated with inflammatory bowel disease (IBD). Diagnosis, especially at an early stage, is difficult and to date there is no diagnostic biomarker. The present study aimed to assess the diagnostic potential of volatile organic compounds (VOCs) in exhaled breath to detect (early) PSC in an IBD population. Methods: Breath samples were obtained from 16 patients with PSC alone, 47 with PSC and IBD, and 53 with IBD alone during outpatient clinic visits. Breath sampling was performed using the ReCIVA breath sampler and subsequently analysed by gas chromatography mass spectrometry. Random forest modelling was performed to find discriminatory VOCs and create a predictive model that was tested using an independent test set. Results: The final model to discriminate patients with PSC, with or without IBD, from patients with IBD alone included twenty VOCs and achieved a sensitivity, specificity, and area under the receiver-operating curve on the test set of 77%, 83%, and 0.84 respectively. Three VOCs (isoprene, 2-octanone and undecane) together correlated significantly with the Amsterdam-Oxford score for PSC disease prognosis. A sensitivity analysis showed stable results across early-stage PSC, including in those with normal alkaline phosphatase levels, as well as further progressed PSC. Conclusion: The present study demonstrates that exhaled breath can distinguish PSC cases from IBD and has potential as a non-invasive clinical breath test for (early) PSC. Impact and implications: Primary sclerosing cholangitis is a complex chronic liver disease, which ultimately results in cirrhosis, liver failure, and death. Detection, especially in early disease stages, can be challenging, and therefore therapy typically starts when there is already some irreversible damage. The current study shows that metabolites in exhaled breath, so called volatile organic compounds, hold promise to non-invasively detect primary sclerosing cholangitis, including at early disease stages.