Low-Density Granulocytes Are a Novel Immunopathological Feature in Both Multiple Sclerosis and Neuromyelitis Optica Spectrum Disorder

Objective: To investigate whether low-density granulocytes (LDGs) are an immunophenotypic feature of patients with multiple sclerosis (MS) or neuromyelitis optica spectrum disorder (NMOSD). Methods: Blood samples were collected from 20 patients with NMOSD and 17 patients with MS, as well as from 15 patients with Systemic Lupus Erythematosus (SLE) and 23 Healthy Donors (HD). We isolated peripheral blood mononuclear cells (PBMCs) with density gradient separation and stained the cells with antibodies against CD14, CD15, CD16, and CD45, and analyzed the cells by flow cytometry or imaging flow cytometry. We defined LDGs as CD14-CD15(high) and calculated their share in total PBMC leukocytes (CD45+) as well as the share of CD16(hi) LDGs. Clinical data on disease course, medication, and antibody status were obtained. Results: LDGs were significantly more common in MS and NMOSD than in HDs, comparable to SLE samples (median values HD 0.2%, MS 0.9%, NMOSD 2.1%, SLE 4.3%). 0/23 of the HDs, but 17/20 NMOSD and 11/17 MS samples as well as 13/15 SLE samples had at least 0.7 % LDGs. NMOSD patients without continuous immunosuppressive treatment had significantly more LDGs compared to their treated counterparts. LDG nuclear morphology ranged from segmented to rounded, suggesting a heterogeneity within the group. Conclusion: LDGs are a feature of the immunophenotype in some patients with MS and NMOSD

Phasor-Based Endogenous NAD(P)H Fluorescence Lifetime Imaging Unravels Specific Enzymatic Activity of Neutrophil Granulocytes Preceding NETosis

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions

Phasor-Based Endogenous NAD(P)H Fluorescence Lifetime Imaging Unravels Specific Enzymatic Activity of Neutrophil Granulocytes Preceding NETosis

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions

Blood Pressure and Same-Day Exposure to Air Pollution at School: Associations with Nano-Sized to Coarse PM in Children

Ultrafine particles (UFP) may contribute to the cardiovascular effects of particulate air pollution, partly because of their relatively efficient alveolar deposition. In this study, we assessed associations between blood pressure and short-term exposure to air pollution in a population of school children.status: publishe

Blood pressure and same-day exposure to air pollution at school : associations with nano-sized to coarse PM in children

BACKGROUND: Ultrafine particles (UFP) may contribute to the cardiovascular effects of particulate air pollution, partly because of their relatively efficient alveolar deposition. OBJECTIVE: In this study, we assessed associations between blood pressure and short-term exposure to air pollution in a population of schoolchildren. METHODS: In 130 children (6–12 years of age), blood pressure was determined during two periods (spring and fall 2011). We used mixed models to study the association between blood pressure and ambient concentrations of particulate matter and ultrafine particles measured in the schools’ playground. RESULTS: Independent of sex, age, height, and weight of the child, parental education, neighborhood socioeconomic status, fish consumption, heart rate, school, day of the week, season, wind speed, relative humidity, and temperature on the morning of examination, an interquartile range (860 particles/cm(3)) increase in nano-sized UFP fraction (20–30 nm) was associated with a 6.35 mmHg (95% CI: 1.56, 11.14; p = 0.01) increase in systolic blood pressure. For the total UFP fraction, systolic blood pressure was 0.79 mmHg (95% CI: 0.07, 1.51; p = 0.03) higher, but no effects on systolic blood pressure were found for the nano-sized fractions with a diameter > 100 nm, nor PM(2.5), PM(coarse), and PM(10). Diastolic blood pressure was not associated with any of the studied particulate mass fractions. CONCLUSION: Children attending school on days with higher UFP concentrations (diameter < 100 nm) had higher systolic blood pressure. The association was dependent on UFP size, and there was no association with the PM(2.5) mass concentration. CITATION: Pieters N, Koppen G, Van Poppel M, De Prins S, Cox B, Dons E, Nelen V, Int Panis L, Plusquin M, Schoeters G, Nawrot TS. 2015. Blood pressure and same-day exposure to air pollution at school: associations with nano-sized to coarse PM in children. Environ Health Perspect 123:737–742; http://dx.doi.org/10.1289/ehp.140812

Decreased mitochondrial DNA content in association with exposure to polycyclic aromatic hydrocarbons in house dust during wintertime: from a population enquiry to cell culture.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants that are formed in combustion processes. At the cellular level, exposure to PAHs causes oxidative stress and/or some of it congeners bind to DNA, which may interact with mitochondrial function. However, the influence of these pollutants on mitochondrial DNA (mtDNA) content remains largely unknown. We determined whether indoor exposure to PAHs is associated with mitochondrial damage as represented by blood mtDNA content. Blood mtDNA content (ratio mitochondrial/nuclear DNA copy number) was determined by real-time qPCR in 46 persons, both in winter and summer. Indoor PAH exposure was estimated by measuring PAHs in sedimented house dust, including 6 volatile PAHs and 8 non-volatile PAHs. Biomarkers of oxidative stress at the level of DNA and lipid peroxidation were measured. In addition to the epidemiologic enquiry, we exposed human TK6 cells during 24 h at various concentrations (range: 0 to 500 µM) of benzo(a)pyrene and determined mtDNA content. Mean blood mtDNA content averaged (± SD) 0.95 ± 0.185. The median PAH content amounted 554.1 ng/g dust (25(th)-75(th) percentile: 390.7-767.3) and 1385 ng/g dust (25(th)-75(th) percentile: 1000-1980) in winter for volatile and non-volatile PAHs respectively. Independent for gender, age, BMI and the consumption of grilled meat or fish, blood mtDNA content decreased by 9.85% (95% CI: -15.16 to -4.2; p = 0.002) for each doubling of non-volatile PAH content in the house dust in winter. The corresponding estimate for volatile PAHs was -7.3% (95% CI: -13.71 to -0.42; p = 0.04). Measurements of oxidative stress were not correlated with PAH exposure. During summer months no association was found between mtDNA content and PAH concentration. The ability of benzo(a)pyrene (range 0 µM to 500 µM) to lower mtDNA content was confirmed in vitro in human TK6 cells. Based on these findings, mtDNA content can be a target of PAH toxicity in humans

GLI2 promoter hypermethylation in saliva of children with a respiratory allergy

Abstract Background The prevalence of respiratory allergy in children is increasing. Epigenetic DNA methylation changes are plausible underlying molecular mechanisms. Results Saliva samples collected in substudies of two longitudinal birth cohorts in Belgium (FLEHS1 & FLEHS2) were used to discover and confirm DNA methylation signatures that can differentiate individuals with respiratory allergy from healthy subjects. Genome-wide analysis with Illumina Methylation 450K BeadChips revealed 23 differentially methylated gene regions (DMRs) in saliva from 11y old allergic children (N=26) vs. controls (N=20) in FLEHS1. A subset of 7 DMRs was selected for confirmation by iPLEX MassArray analysis. First, iPLEX analysis was performed in the same 46 FLEHS1 samples for analytical confirmation of the findings obtained during the discovery phase. iPLEX results correlated significantly with the 450K array data (P <0.0001) and confirmed 4 out of the 7 DMRs. Aiming for additional biological confirmation, the 7 DMRs were analyzed using iPLEX in a substudy of an independent birth cohort (FLEHS2; N=19 cases vs. 20 controls, aged 5 years). One DMR in the GLI2 promoter region showed a consistent statistically significant hypermethylation in individuals with respiratory allergy across the two birth cohorts and technologies. In addition to its involvement in TGF-β signaling and T-helper differentiation, GLI2 has a regulating role in lung development. Conclusion GLI2 is considered an interesting candidate DNA methylation marker for respiratory allergy