Investigating the impact of London's ultra low emission zone on children's health: children's health in London and Luton (CHILL) protocol for a prospective parallel cohort study.

BACKGROUND: Air pollution harms health across the life course. Children are at particular risk of adverse effects during development, which may impact on health in later life. Interventions that improve air quality are urgently needed both to improve public health now, and prevent longer-term increased vulnerability to chronic disease. Low Emission Zones are a public health policy intervention aimed at reducing traffic-derived contributions to urban air pollution, but evidence that they deliver health benefits is lacking. We describe a natural experiment study (CHILL: Children's Health in London and Luton) to evaluate the impacts of the introduction of London's Ultra Low Emission Zone (ULEZ) on children's health. METHODS: CHILL is a prospective two-arm parallel longitudinal cohort study recruiting children at age 6-9 years from primary schools in Central London (the focus of the first phase of the ULEZ) and Luton (a comparator site), with the primary outcome being the impact of changes in annual air pollutant exposures (nitrogen oxides [NOx], nitrogen dioxide [NO2], particulate matter with a diameter of less than 2.5micrograms [PM2.5], and less than 10 micrograms [PM10]) across the two sites on lung function growth, measured as post-bronchodilator forced expiratory volume in one second (FEV1) over five years. Secondary outcomes include physical activity, cognitive development, mental health, quality of life, health inequalities, and a range of respiratory and health economic data. DISCUSSION: CHILL's prospective parallel cohort design will enable robust conclusions to be drawn on the effectiveness of the ULEZ at improving air quality and delivering improvements in children's respiratory health. With increasing proportions of the world's population now living in large urban areas exceeding World Health Organisation air pollution limit guidelines, our study findings will have important implications for the design and implementation of Low Emission and Clean Air Zones in the UK, and worldwide. CLINICALTRIALS: GOV: NCT04695093 (05/01/2021).

Device-Measured Change in Physical Activity in Primary School Children During the UK COVID-19 Pandemic Lockdown: A Longitudinal Study.

BACKGROUND: Lockdown measures, including school closures, due to the COVID-19 pandemic have caused widespread disruption to children's lives. The aim of this study was to explore the impact of a national lockdown on children's physical activity using seasonally matched accelerometry data. METHODS: Using a pre/post observational design, 179 children aged 8 to 11 years provided physical activity data measured using hip-worn triaxial accelerometers worn for 5 consecutive days prepandemic and during the January to March 2021 lockdown. Multilevel regression analyses adjusted for covariates were used to assess the impact of lockdown on time spent in sedentary and moderate to vigorous physical activity. RESULTS: A 10.8-minute reduction in daily time spent in moderate to vigorous physical activity (standard error: 2.3 min/d, P < .001) and a 33.2-minute increase in daily sedentary activity (standard error: 5.5 min/d, P < .001) were observed during lockdown. This reflected a reduction in daily moderate to vigorous physical activity for those unable to attend school (-13.1 [2.3] min/d, P < .001) during lockdown, with no significant change for those who continued to attend school (0.4 [4.0] min/d, P < .925). CONCLUSION: These findings suggest that the loss of in-person schooling was the single largest impact on physical activity in this cohort of primary school children in London, Luton, and Dunstable, United Kingdom.

E-cigarette vapour enhances pneumococcal adherence to airway epithelial cells.

E-cigarette vapour contains free radicals with the potential to induce oxidative stress. Since oxidative stress in airway cells increases platelet-activating factor receptor (PAFR) expression, and PAFR is co-opted by pneumococci to adhere to host cells, we hypothesised that E-cigarette vapour increases pneumococcal adhesion to airway cells.Nasal epithelial PAFR was assessed in non-vaping controls, and in adults before and after 5 min of vaping. We determined the effect of vapour on oxidative stress-induced, PAFR-dependent pneumococcal adhesion to airway epithelial cells in vitro, and on pneumococcal colonisation in the mouse nasopharynx. Elemental analysis of vapour was done by mass spectrometry, and oxidative potential of vapour assessed by antioxidant depletion in vitroThere was no difference in baseline nasal epithelial PAFR expression between vapers (n=11) and controls (n=6). Vaping increased nasal PAFR expression. Nicotine-containing and nicotine-free E-cigarette vapour increased pneumococcal adhesion to airway cells in vitro Vapour-stimulated adhesion in vitro was attenuated by the PAFR blocker CV3988. Nicotine-containing E-cigarette vapour increased mouse nasal PAFR expression, and nasopharyngeal pneumococcal colonisation. Vapour contained redox-active metals, had considerable oxidative activity, and adhesion was attenuated by the antioxidant N-acetyl cysteine.This study suggests that E-cigarette vapour has the potential to increase susceptibility to pneumococcal infection.

Childhood victimization and inflammation in young adulthood: A genetically sensitive cohort study.

OBJECTIVE: Childhood victimization is an important risk factor for later immune-related disorders. Previous evidence has demonstrated that childhood victimization is associated with elevated levels of inflammation biomarkers measured decades after exposure. However, it is unclear whether this association is (1) already detectable in young people, (2) different in males and females, and (3) confounded by genetic liability to inflammation. Here we sought to address these questions. METHOD: Participants were 2232 children followed from birth to age 18years as part of the Environmental Risk (E-Risk) Longitudinal Twin Study. Childhood victimization was measured prospectively from birth to age 12years. Inflammation was measured through C-reactive protein (CRP) levels in dried blood spots at age 18years. Latent genetic liability for high inflammation levels was assessed through a twin-based method. RESULTS: Greater exposure to childhood victimization was associated with higher CRP levels at age 18 (serum-equivalent means were 0.65 in non-victimized Study members, 0.74 in those exposed to one victimization type, and 0.81 in those exposed to poly-victimization; p=0.018). However, this association was driven by a significant association in females (serum-equivalent means were 0.75 in non-victimized females, 0.87 in those exposed to one type of victimization, and 1.19 in those exposed to poly-victimization; p=0.010), while no significant association was observed in males (p=0.19). Victimized females showed elevated CRP levels independent of latent genetic influence, as well as childhood socioeconomic status, and waist-hip ratio and body temperature at the time of CRP assessment. CONCLUSION: Childhood victimization is associated with elevated CRP levels in young women, independent of latent genetic influences and other key risk factors. These results strengthen causal inference about the effects of childhood victimization on inflammation levels in females by accounting for potential genetic confounding.

Urban particulate matter stimulation of human dendritic cells enhances priming of naive CD8 T lymphocytes.

Epidemiological studies have consistently shown associations between elevated concentrations of urban particulate matter (UPM) air pollution and exacerbations of asthma and chronic obstructive pulmonary disease, which are both associated with viral respiratory infections. The effects of UPM on dendritic cell (DC) -stimulated CD4 T lymphocytes have been investigated previously, but little work has focused on CD8 T-lymphocyte responses despite their importance in anti-viral immunity. To address this, we examined the effects of UPM on DC-stimulated naive CD8 T-cell responses. Expression of the maturation/activation markers CD83, CCR7, CD40 and MHC class I on human myeloid DCs (mDCs) was characterized by flow cytometry after stimulation with UPMin vitro in the presence/absence of granulocyte-macrophage colony-stimulating factor (GM-CSF). The capacity of these mDCs to stimulate naive CD8 T-lymphocyte responses in allogeneic co-culture was then assessed by measuring T-cell cytokine secretion using cytometric bead array, and proliferation and frequency of interferon-γ (IFN-γ)-producing T lymphocytes by flow cytometry. Treatment of mDCs with UPM increased expression of CD83 and CCR7, but not MHC class I. In allogeneic co-cultures, UPM treatment of mDCs enhanced CD8 T-cell proliferation and the frequency of IFN-γ+ cells. The secretion of tumour necrosis factor-α, interleukin-13, Granzyme A and Granzyme B were also increased. GM-CSF alone, and in concert with UPM, enhanced many of these T-cell functions. The PM-induced increase in Granzyme A was confirmed in a human experimental diesel exposure study. These data demonstrate that UPM treatment of mDCs enhances priming of naive CD8 T lymphocytes and increases production of pro-inflammatory cytokines. Such UPM-induced stimulation of CD8 cells may potentiate T-lymphocyte cytotoxic responses upon concurrent airway infection, increasing bystander damage to the airways.

Cigarette smoke-induced induction of antioxidant enzyme activities in airway leukocytes is absent in active smokers with COPD.

BACKGROUND: Oxidative injury to the airway has been proposed as an important underlying mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). As the extent of oxidant-mediated damage is dependent on the endogenous antioxidant defences within the airways, we examined whether COPD was associated with deficiencies in the antioxidant network within the respiratory tract lining fluids (RTLFs) and resident airway leukocytes. We hypothesised that COPD would be associated with both basal depression of antioxidant defences and impaired adaptive antioxidant responses to cigarette smoke. METHODS: Low molecular weight and enzymatic antioxidants together with metal-handling proteins were quantified in bronchoalveolar lavage fluid and airway leukocytes, derived from current (n=9) and ex-smoking COPD patients (n=15), as well as from smokers with normal lung function (n=16) and healthy never smokers (n=13). RESULTS: Current cigarette smoking was associated with an increase in ascorbate and glutathione within peripheral RTLFs in both smokers with normal lung function compared with healthy never smokers and in COPD smokers compared with COPD ex-smokers. In contrast, intra-cellular antioxidant enzyme activities (glutathione peroxidase, glutathione reductase, and catalase) were only up-regulated in smokers with normal lung function compared with healthy never smokers and not in actively smoking COPD patients relative to COPD ex-smokers. CONCLUSIONS: We found no evidence of impaired basal antioxidant defences, within either the RTLFs or airway leukocytes in stable ex-smoking COPD patients compared with healthy never smoking controls. Current cigarette smoking induced an up-regulation of low molecular weight antioxidants in the RTLFs of both control subjects with normal lung function and patients with COPD. Importantly, the present data demonstrated a cigarette smoke-induced increase in intra-cellular antioxidant enzyme activities only within the smokers with normal lung function, implying that patients with COPD who continue to smoke will experience enhanced oxidative stress, prompting disease progression.

Comparative gene expression profiling of human umbilical vein endothelial cells and ocular vascular endothelial cells.

BACKGROUND/AIMS: To investigate the difference between human umbilical vein endothelial cells (HUVEC) and human ocular microvascular endothelial cell (MVEC) gene expression, and to determine if these differences could improve the understanding of ocular angiogenic diseases. METHODS: The gene expression profiles of HUVEC and matched unpassaged human choroidal, retinal and iris endothelial cells were conducted using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Selected differences were confirmed by real time PCR. Functional cell proliferation assays were used to support microarray findings. RESULTS: HUVEC showed enrichment for probe sets involved in embryological development while ocular MVEC demonstrated enrichment for probe sets for MHC classes I and II, immune responses and cell signal transduction. Comparison of human retinal and choroidal endothelial cells demonstrated significant differences in the expression of probe sets encoding insulin-like growth factor 1 (IGF-1) signalling. Cell proliferation assays demonstrated the stimulatory role of IGF-1 on retinal endothelial cells compared with choroidal endothelial cells. CONCLUSIONS: Gene expression profiling has demonstrated that HUVEC are probably not a suitable surrogate for the study of ocular angiogenic disorders. There are also significant differences in the gene expression of human retinal and choroidal endothelial cells, which may be important in the mechanism and treatment of choroidal and retinal neovascularisation.

Proinflammatory doses of diesel exhaust in healthy subjects fail to elicit equivalent or augmented airway inflammation in subjects with asthma.

BACKGROUND: Exposure to traffic-derived air pollutants, particularly diesel emissions, has been associated with adverse health effects, predominantly in individuals with pre-existing respiratory disease. Here the hypothesis that this heightened sensitivity reflects an augmentation of the transient inflammatory response previously reported in healthy adults exposed to diesel exhaust is examined. METHODS: 32 subjects with asthma (mild to moderate severity) and 23 healthy controls were exposed in a double-blinded crossover control fashion to both filtered air and diesel exhaust (100 µg/m(3) PM(10)) for 2 h. Airway inflammation was assessed by bronchoscopy 18 h postexposure. In addition, lung function, fraction of exhaled nitric oxide and bronchial reactivity to metacholine were examined in the subjects with asthma. RESULTS: In healthy control subjects a significant increase in submucosal neutrophils (p=0.004) was observed following the diesel challenge. Significant increases in neutrophil numbers (p=0.01), and in the concentrations of interleukin 6 (p=0.03) and myeloperoxidase (p=0.04), were also seen in bronchial wash after diesel, relative to the control air challenge. No evidence of enhanced airway inflammation was observed in the subjects with asthma following the diesel exposure. CONCLUSIONS: Exposure to diesel exhaust at concentrations consistent with roadside levels elicited an acute and active neutrophilic inflammation in the airways of healthy subjects. This response was absent in subjects with asthma, as was evidence supporting a worsening of allergic airway inflammation.

Antioxidant airway responses following experimental exposure to wood smoke in man.

BACKGROUND: Biomass combustion contributes to the production of ambient particulate matter (PM) in rural environments as well as urban settings, but relatively little is known about the health effects of these emissions. The aim of this study was therefore to characterize airway responses in humans exposed to wood smoke PM under controlled conditions. Nineteen healthy volunteers were exposed to both wood smoke, at a particulate matter (PM2.5) concentration of 224 ± 22 µg/m3, and filtered air for three hours with intermittent exercise. The wood smoke was generated employing an experimental set-up with an adjustable wood pellet boiler system under incomplete combustion. Symptoms, lung function, and exhaled NO were measured over exposures, with bronchoscopy performed 24 h post-exposure for characterisation of airway inflammatory and antioxidant responses in airway lavages. RESULTS: Glutathione (GSH) concentrations were enhanced in bronchoalveolar lavage (BAL) after wood smoke exposure vs. air (p = 0.025), together with an increase in upper airway symptoms. Neither lung function, exhaled NO nor systemic nor airway inflammatory parameters in BAL and bronchial mucosal biopsies were significantly affected. CONCLUSIONS: Exposure of healthy subjects to wood smoke, derived from an experimental wood pellet boiler operating under incomplete combustion conditions with PM emissions dominated by organic matter, caused an increase in mucosal symptoms and GSH in the alveolar respiratory tract lining fluids but no acute airway inflammatory responses. We contend that this response reflects a mobilisation of GSH to the air-lung interface, consistent with a protective adaptation to the investigated wood smoke exposure.

Can vitamin C induce nucleotide excision repair? Support from in vitro evidence.

Intracellular vitamin C acts to protect cells against oxidative stress by intercepting reactive oxygen species (ROS) and minimising DNA damage. However, rapid increases in intracellular vitamin C may induce ROS with subsequent DNA damage priming DNA repair processes. Herein, we examine the potential of vitamin C and the derivative ascorbate-2-phosphate (2-AP) to induce a nucleotide excision repair (NER) response to DNA damage in a model of peripheral blood mononuclear cells. Exposure of cells to elevated levels of vitamin C induced ROS activity, resulting in increased levels of deoxycytidine glyoxal (gdC) and 8-oxo-2'-deoxyguanosine (8-oxodG) adducts in DNA; a stress response was also induced by 2-AP, but was delayed in comparison to vitamin C. Evidence of gdC repair was also apparent. Measurement of cyclobutane thymine-thymine dimers (T < >T) in DNA and culture supernatant were included as a positive marker for NER activity; this was evidenced by a reduction in DNA and increases in culture supernatant levels of T < >T for vitamin C-treated cells. Genomics analysis fully supported these findings confirming that 2-AP, in particular, induced genes associated with stress response, cell cycle arrest, DNA repair and apoptosis, and additionally provided evidence for the involvement of vitamin C in the mobilisation of intracellular catalytic Fe.

Detection of choline transporter-like 1 protein CTL1 in neuroblastoma x glioma cells and in the CNS, and its role in choline uptake.

Choline is an essential nutrient necessary for synthesis of membrane phospholipids, cell signalling molecules and acetylcholine. The aim of this study was to detect and characterize the choline transporter-like 1 (CTL1/SLC44A1) protein in CNS tissues and the hybrid neuroblastoma x glioma cell line NG108-15, which synthesizes acetylcholine and has high affinity choline transport but does not express the cholinergic high affinity choline transporter 1. The presence of CTL1 protein in NG108-15 cells was confirmed using our antibody G103 which recognizes the C-terminal domain of human CTL1. Three different cognate small interfering RNAs were used to decrease CTL1 mRNA in NG108-15 cells, causing lowered CTL1 protein expression, choline uptake and cell growth. None of the small interfering RNAs influenced carnitine transport, demonstrating the absence of major non-specific effects. In parental C6 cells knockdown of CTL1 also reduced high affinity choline transport. Our results support the concept that CTL1 protein is necessary for the high affinity choline transport which supplies choline for cell growth. The presence of CTL1 protein in rat and human CNS regions, where it is found in neuronal, glial and endothelial cells, suggests that malfunction of this transporter could have important implications in nervous system development and repair following injury, and in neurodegenerative diseases.

Glutamate receptor expression in multiple sclerosis lesions.

Blockade of receptors for the excitatory neurotransmitter glutamate ameliorates neurological clinical signs in models of the CNS inflammatory demyelinating disease multiple sclerosis (MS). To investigate whether glutamate excitoxicity may play a role in MS pathogenesis, the cellular localization of glutamate and its receptors, transporters and enzymes was examined. Expression of glutamate receptor (GluR) 1, a Ca(++)-permeable ionotropic AMPA receptor subunit, was up-regulated on oligodendrocytes in active MS lesion borders, but Ca(++)-impermeable AMPA GluR2 subunit levels were not increased. Reactive astrocytes in active plaques expressed AMPA GluR3 and metabotropic mGluR1, 2/3 and 5 receptors and the GLT-1 transporter, and a subpopulation was immunostained with glutamate antibodies. Activated microglia and macrophages were immunopositive for GluR2, GluR4 and NMDA receptor subunit 1. Kainate receptor GluR5-7 immunostaining showed endothelial cells and dystrophic axons. Astrocyte and macrophage populations expressed glutamate metabolizing enzymes and unexpectedly the EAAC1 transporter, which may play a role in glutamate uptake in lesions. Thus, reactive astrocytes in MS white matter lesions are equipped for a protective role in sequestering and metabolizing extracellular glutamate. However, they may be unable to maintain glutamate at levels low enough to protect oligodendrocytes rendered vulnerable to excitotoxic damage because of GluR1 up-regulation.

A comparison of the gene expression profiles of CRL-1807 colonocytes exposed to endogenous AAPH-generated peroxides and exogenous peroxides from heated oil.

Oxidation of PUFAs in the diet has the potential to be genotoxic and hence carcinogenic. Such carcinogenic processes originate within stem cells of the colon. These cells appear to be predisposed to the carcinogenic process. In colon cells (CRL-1807) exposed to chemical reactions simulating exogenous and endogenous peroxidation reactions, we have observed that undifferentiated cells could mount an effective recombinational repair/TCR response to an endogenous peroxidative DNA damage insult, but not to an external exogenous peroxidative insult as one would encounter from a dietary source. This may suggest that defects in such specific DNA repair may play a role in tumour development in undifferentiated colonocytes exposed to a diet-derived lipid peroxides.

Evidence for attenuated cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine removal in cancer patients.

Measurement of the products of oxidatively damaged DNA in urine is a frequently used means by which oxidative stress may be assessed non-invasively. We believe that urinary DNA lesions, in addition to being biomarkers of oxidative stress, can potentially provide more specific information, for example, a reflection of repair activity. We used high-performance liquid chromatography prepurification, with gas chromatography-mass spectrometry (LC-GC-MS) and ELISA to the analysis of a number of oxidative [e.g., 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-guanine, 5-(hydroxymethyl)uracil], non-oxidative (cyclobutane thymine dimers) and oligomeric DNA products in urine. We analysed spot urine samples from 20 healthy subjects, and 20 age- and sex-matched cancer patients. Mononuclear cell DNA 8-oxodG levels were assessed by LC-EC. The data support our proposal that urinary DNA lesion products are predominantly derived from DNA repair. Furthermore, analysis of DNA and urinary 8-oxodG in cancer patients and controls suggested reduced repair activity towards this lesion marker in these patients.

DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine.

The repair of oxidatively damaged DNA is integral to the maintenance of genomic stability, and hence prevention of a wide variety of pathological conditions, such as aging, cancer and cardiovascular disease. The ability to non-invasively assess DNA repair may provide information regarding repair pathways, variability in repair capacity, and susceptibility to disease. The development of assays to measure urinary DNA lesions offered this potential, although it rapidly became clear that possible contribution from diet and cell turnover may influence urinary lesion levels. Whilst early studies attempted to address these issues, up until now, much of the data appears conflicting. However, recent work from our laboratories, in which human volunteers were fed highly oxidatively modified 15N-labelled DNA demonstrates that diet does not appear to contribute to urinary levels of 8-hydroxyguanine and 7,8-dihydro-8-oxo-2'-deoxyguanosine. Furthermore, we propose that a number of literature reports form an argument against a contribution from cell death. Indeed we, and others, have presented evidence, which strongly suggests the involvement of cell death to be minimal. Taken together, these data would appear to rule out various confounding factors, leaving DNA repair pathways as the principal source of urinary purine, if not DNA, lesions enabling such measurements to be used as indicators of repair.