The utility of a standardised breath sampler in school age children within a real-world prospective study.

Clinical assessment of children with asthma is problematic, and non-invasive biomarkers are needed urgently. Monitoring exhaled volatile organic compounds (VOCs) is an attractive alternative to invasive tests (blood and sputum) and may be used as frequently as required. Standardised reproducible breath-sampling is essential for exhaled-VOC analysis, and although the ReCIVA (Owlstone Medical Limited) breath-sampler was designed to satisfy this requirement, paediatric use was not in the original design brief. The efficacy of the ReCIVA at sampling breath from children has been studied, and 90 breath-samples from 64 children (5-15 years) with, and without asthma (controls), were collected with two different ReCIVA units. Seventy samples (77.8%) contained the specified 1 l of sampled-breath. Median sampling times were longer in children with acute asthma (770.2 s, range: 532.2-900.1 s) compared to stable asthma (690.6 s, range: 477.5-900.1 s;p= 0.01). The ReCIVA successfully detected operational faults, in 21 samples. A leak, caused by a poor fit of the face mask seal was the most common (15); the others were USB communication-faults (5); and, a single instance of a file-creation error. Paediatric breath-profiles were reliably monitored, however synchronisation of sampling to breathing-phases was sometimes lost, causing some breaths not to be sampled, and some to be sampled continuously. This occurred in 60 (66.7%) of the samples and was a source of variability. Importantly, multi-variate modelling of untargeted VOC analysis indicated the absence of significant batch effects for eight operational variables. The ReCIVA appears suitable for paediatric breath-sampling. Post-processing of breath-sample meta-data is recommended to assess the quality of sample-acquisition. Further, future studies should explore the effect of pump-synchronisation faults on recovered VOC profiles, and mask sizes to fit all ages will reduce the potential for leaks and importantly, provide higher levels of comfort to children with asthma.

Reducing Asthma Attacks in Children using Exhaled Nitric Oxide as a biomarker to inform treatment strategy: a randomised trial (RAACENO).

BACKGROUND: Childhood asthma is a common condition. Currently there is no validated objective test which can be used to guide asthma treatment in children. This study tests the hypothesis that the addition of fractional exhaled nitric oxide (FENO) monitoring in addition to standard care reduces the number of exacerbations (or attacks) in children with asthma. METHODS: This is a multi-centre, randomised controlled study. Children will be included of age 6-16 years who have a diagnosis of asthma, currently use inhaled corticosteroids (ICSs) and have had an exacerbation in the previous 12 months. Exclusion criteria include being unable to provide FENO measurement at baseline assessment, having another chronic respiratory condition and being currently treated with maintenance oral steroids. Participants will be recruited in both primary and secondary care settings and will be randomised to either receive asthma treatment guided by FENO plus symptoms (FENO group) or asthma treatment guided by symptoms only (standard care group). Within the FENO group, different treatment decisions will be made dependent on changes in FENO. Participants will attend assessments 3, 6, 9 and 12 months post randomisation. The primary outcome is asthma exacerbation requiring prescription and/or use of an oral corticosteroid over 12 months as recorded by the participant/parent or in general practitioner records. Secondary outcomes include time to first attack, number of attacks, asthma control score and quality of life. Adherence to ICS treatment is objectively measured by an electronic logging device. Participants are invited to participate in a "phenotyping" assessment where skin prick reactivity and bronchodilator response are determined and a saliva sample is collected for DNA extraction. Qualitative interviews will be held with participants and research nurses. A health economic evaluation will take place. DISCUSSION: This study will evaluate whether FENO can provide an objective index to guide and stratify asthma treatment in children. TRIAL REGISTRATION: ISRCTN, ISRCTN67875351. Registered on 12 April 2017. Prospectively registered.

The independent role of prenatal and postnatal exposure to active and passive smoking on the development of early wheeze in children.

Maternal smoking during pregnancy increases childhood asthma risk, but health effects in children of nonsmoking mothers passively exposed to tobacco smoke during pregnancy are unclear. We examined the association of maternal passive smoking during pregnancy and wheeze in children aged ≤2 years.Individual data of 27 993 mother-child pairs from 15 European birth cohorts were combined in pooled analyses taking into consideration potential confounders.Children with maternal exposure to passive smoking during pregnancy and no other smoking exposure were more likely to develop wheeze up to the age of 2 years (OR 1.11, 95% CI 1.03-1.20) compared with unexposed children. Risk of wheeze was further increased by children's postnatal passive smoke exposure in addition to their mothers' passive exposure during pregnancy (OR 1.29, 95% CI 1.19-1.40) and highest in children with both sources of passive exposure and mothers who smoked actively during pregnancy (OR 1.73, 95% CI 1.59-1.88). Risk of wheeze associated with tobacco smoke exposure was higher in children with an allergic versus nonallergic family history.Maternal passive smoking exposure during pregnancy is an independent risk factor for wheeze in children up to the age of 2 years. Pregnant females should avoid active and passive exposure to tobacco smoke for the benefit of their children's health.

Blood eosinophils as a marker of likely corticosteroid response in children with preschool wheeze: time for an eosinophil guided clinical trial?

Childhood wheezing is common particularly in children under the age of 6 years and in this age group is generally referred to as preschool wheezing. Particular diagnostic and treatment uncertainties exist in these young children due to the difficulty in obtaining objective evidence of reversible airways narrowing and inflammation. A diagnosis of asthma depends on the presence of relevant clinical signs and symptoms and the demonstration of reversible airways narrowing on lung function testing, which is difficult to perform in young children. Few treatments are available and inhaled corticosteroids are the recommended preventer treatment in most international asthma guidelines. There is, however, considerable controversy about its effectiveness in children with preschool wheeze and a corticosteroid responder phenotype has not been established. These diagnostic and treatment uncertainties in conjunction with the knowledge of corticosteroid side effects, in particular the reduction of growth velocity, have resulted in a variable approach to inhaled corticosteroid prescribing by medical practitioners and a reluctance in carers to regularly administer the treatment. Identifying children who are likely responders to corticosteroid therapy would be a major benefit in the management of this condition. Eosinophils have emerged as a promising biomarker of corticosteroid responsive airways disease, and evaluation of this biomarker in sputum has successfully been employed to direct management in adults with asthma. Obtaining sputum from young children is time consuming and difficult, and it is hard to justify more invasive procedures such as a bronchoscopy in young children routinely. Recently, in children, interest has shifted to assessing the value of less invasive biomarkers of likely corticosteroid response and the biomarker 'blood eosinophils' has emerged as an attractive candidate. The aim of this review was to summarize the evidence for blood eosinophils as a predictive biomarker for corticosteroid responsive disease with a particular focus on the difficult area of preschool wheeze.

Therapeutic vascular compliance change may cause significant variation in coronary perfusion: a numerical study.

In some pathological conditions like aortic stiffening and calcific aortic stenosis (CAS), the microstructure of the aortic root and the aortic valve leaflets are altered in response to stress resulting in changes in tissue thickness, stiffness, or both. This aortic stiffening and CAS are thought to affect coronary blood flow. The goal of the present paper was to include the flow in the coronary ostia in the previous fluid structure interaction model we have developed and to analyze the effect of diseased tissues (aortic root stiffening and CAS) on coronary perfusion. Results revealed a significant impact on the coronary perfusion due to a moderate increase in the aortic wall stiffness and CAS (increase of the aortic valve leaflets thickness). A marked drop of coronary peak velocity occurred when the values of leaflet thickness and aortic wall stiffness were above a certain threshold, corresponding to a threefold of their normal value. Consequently, mild and prophylactic treatments such as smoking cessation, exercise, or diet, which have been proven to increase the aortic compliance, may significantly improve the coronary perfusion.

Early detection of age-related memory deficits in individual mice.

To date, no consensus has been reached concerning the age of the earliest onset of age-related cognitive deficits in rodents. Our aim was to develop a behavioral model allowing early and individual detection of age-related cognitive impairments. We tested young (3 months), middle-aged (10 months) and aged (17 months) C57Bl/6 mice in the starmaze, a task allowing precise analysis of the search pattern of mice via standardized calculation of two navigation indices. We performed mouse-per-mouse analyses and compared each mouse's performance to a threshold based on young mice's performances. Using this method we identified impaired mice from the age of 10 months old. Their deficits were independent of any sensorimotor dysfunctions and were associated with an alteration of the maintenance of the hippocampal CA1 late-LTP. This study develops reliable methodology for early detection of age-related memory disorders and provides evidence that memory can decline in some individuals as early as from the age of 10 months.

The moisturizing effect of a wheat extract food supplement on women's skin: a randomized, double-blind placebo-controlled trial.

Ceramides, specific lipid components of the skin, represent 35-40% of the intercellular cement binding cells together and contributing to skin hydration. A wheat extract rich in ceramides and digalactosyl-diglycerides was developed by Hitex in two forms: wheat extract oil (WEO) and wheat extract powder (WEP). In vitro tests and two clinical studies demonstrated promising efficacy results with WEP on skin hydration. To confirm these early results, a double-blind, randomized, placebo-controlled study was carried out on 51 women aged 20-63 years with dry to very dry skin who received either 350 mg of WEO or placebo for 3 months. Evaluation of skin hydration on legs, arms and face, assessed at baseline (D0) and at study end (D84) was performed by the dermatologist using dermatological scores (dryness, roughness, erythema), skin hydration measurement (corneometry) and self-assessment scores (Visual Analogue Scale: VAS). Perceived efficacy was noted by participants throughout the study; tolerability and overall acceptability of the study products were evaluated by the dermatologist and the participants at the end of study. Skin hydration was significantly increased between D0 and D84 on the arms (P < 0.001) and legs (P = 0.012) in the WEO group compared with placebo. Even if no significant statistical differences between groups were observed for the dermatological evaluation, skin dryness and redness tended to be reduced in the WEO group. Moreover, from D0 to D84, the VAS index had a tendency to increase in favour of WEO for the overall skin hydration (P = 0.084) indicating that participants perceived an improvement. The WEO capsules were perceived by participants as being more effective than placebo on all skin dryness signs. In conclusion, WEO capsules were well tolerated and appreciated. After 3 months' treatment, a significant increase in skin hydration and an improvement in associated clinical signs were observed in women with dry skin.

Compositional studies of human RPE lipofuscin: mechanisms of molecular modifications.

The accumulation of lipofuscin has previously been implicated in several retinal diseases including Best's macular dystrophy, Stargardt's disease and age-related macular degeneration (AMD). Previously one of the major fluorophores of lipofuscin was identified as a bis-retinoid pyridinium salt called A2E, which is known to photochemically cause damage. In addition to A2E, there are numerous components in RPE lipofuscin that are unidentified. These compounds were determined to be structurally related to A2E by their fragmentation pattern with losses of 106, 190, 174 and/or 150 amu from the parent ion and the formation of fragments of ca 592 amu. The vast majority consists of relatively hydrophobic components corresponding to derivatized A2E with molecular weights in discrete groups of 800-900, 970-1080 and > 1200 m/z regions. In order to determine the mechanism of these modifications, A2E was chemically modified by; (1) the formation of specific esters, (2) reaction with specific aldehydes and (3) spontaneous auto-oxidation. The contribution of ester formation to the naturally occurring components of lipofuscin was discounted since their fragmentation patterns were different to those found in vivo. Alternatively, reactions with specific aldehydes result in nearly identical products as those found in vivo. Artificial aging of RPE lipofuscin gives a complex mixture of structurally related components. This results from the auto- and/or photooxidation of A2E to form aldehydes, which then back react with A2E giving a series of higher molecular weight products. The majority of these modifications result in compounds that are much more hydrophobic than A2E. These higher molecular weight materials have increased values of log P compared to A2E. This increase in hydrophobicity most likely aids in the sequestering of A2E into granules with the concomitant diminution of its reactivity. Therefore, these processes may serve as protective mechanisms for the RPE.

Compositional studies of human RPE lipofuscin.

Age-related macular degeneration (AMD) is an ocular disease that causes visual loss and legal blindness in the elderly population. The etiology of AMD is complex and may include genetic predispositions, accumulation of lipofuscin and drusen, local inflammation and neovascularization. The accumulation of lipofuscin has been shown to precede the death of photoreceptor cells and the deterioration of the RPE. As a result, the determination of the photosensitive components of lipofuscin has been of major interest. One of these components, previously identified as a bis-retinoid pyridinium compound, is referred to as A2E. A2E has been characterized by mass spectrometry and is known to have a mass of 592 Da. Most remaining chromophores in RPE lipofuscin are structurally related to A2E as determined by their fragmentation pattern with losses of M ± 190, 174 and/or 150 Da. Analysis of lipofuscin from various donors indicated that the extracts consist of as many as 15 of these hydrophobic components, which are also observed to form spontaneously in vitro over extended periods of time. These consist of ca 90% of the A2E-like components in RPE lipofuscin and correspond to derivatized A2E with discrete molecular weights of 800-900 m/z, 970-1080 m/z and above 1200 m/z regions. It was determined that these species are formed from self-reaction of A2E oxidation products or their reaction with A2E itself to form higher molecular weight products. The majority of modifications are much more hydrophobic than A2E and exhibit increasingly higher values of log P. This acts as a driving force for the sequestering of A2E into granules resulting in a concomitant diminution of its reactivity in vivo.

In vitro investigation of the impact of aortic valve stenosis severity on left coronary artery flow.

Patients with aortic valve stenosis (AS) may experience angina pectoris even if they have angiographically normal coronary arteries. Angina is associated with a marked increase in the risk of sudden death in AS patients. Only a few in vitro models describing the interaction between the left ventricular and aortic pressures, and the coronary circulation have been reported. These models were designed for specific research studies and they need to be improved or modified when other specific studies are required. Consequently, we have developed an in vitro model that is able to mimic the coronary circulation in presence of aortic stenosis. First, we have validated the model under physiological conditions. Then, we have examined and quantified the hemodynamic effects of different degrees of AS (from normal to severe AS) on the coronary flow using a model of the normal left coronary artery. In the coronary in vitro model without AS (normal valve), the amplitude and shape of coronary flow were similar to those observed in in vivo measurements obtained under physiological conditions, as described by Hozumi et al. (1998, "Noninvasive Assessment of Significant Left Anterior Descending Coronary Artery Stenosis by Coronary Flow Velocity Reserve With Transthoracic Color Doppler Echocardiography," Circulation, 97, pp. 1557-1562). The presence of an AS induced an increase in the maximum and mean coronary flow rates (97% and 73%, respectively, for a very severe AS). Furthermore, when AS was very severe, a retrograde flow occurred during systole. This study allowed us to validate our coronary in vitro model under physiological conditions, both in the absence and presence of AS. These changes could explain the fact that even if patients have angiographically normal epicardial coronary arteries, we can observe the occurrence of angina pectoris in these patients in the presence of an AS.

Age-related accumulation of 3-nitrotyrosine and nitro-A2E in human Bruch's membrane.

Age-related macular degeneration (AMD) is a disease leading to severe visual loss and legal blindness in the elderly population. The pathophysiology of AMD is complex and may include genetic predispositions, accumulation of lipofuscin and drusen, local inflammation and neovascularization. Recently four independent research groups have identified a commonly inherited variant (Y402H) of the complement factor H gene in the genome from different groups of AMD patients. The Y402H variant of CFH significantly increases the risk of AMD and links the genetics of the disease with inflammation. During inflammation there is activation of inducible nitric oxide synthase and release of nitric oxide, which in principal could lead to non-enzymatic nitration within extracellular deposits and/or intrinsic extracellular matrix protein components of human Bruch's membrane. We have identified two biomarkers for non-enzymatic nitration in aged human Bruch's membrane, indicative of inflammation, that include 3-nitrotyrosine identified in Bruch's membrane preparations and nitrated A2E from the lipid soluble extract of the Bruch's membrane preparation. Approximately 30-40 times more A2E is observed in samples of the organic soluble extract of lipofuscin compared to the extract of Bruch's membrane. It is of interest to note that although A2E is a major constituent of RPE lipofuscin, nitrated A2E could not be detected in RPE extracts. We show here that nitro-A2E is a specific biomarker of nitrosative stress in Bruch's membrane and its concentration correlates directly with tissue age.

Optimization of Doppler velocity echocardiographic measurements using an automatic contour detection method.

Intra- and inter-observer variability in Doppler velocity echocardiographic measurements (DVEM) is a significant issue. Indeed, imprecisions of DVEM can lead to diagnostic errors, particularly in the quantification of the severity of heart valve dysfunction. To minimize the variability and rapidity of DVEM, we have developed an automatic method of Doppler velocity wave contour detection, based on active contour models. To validate our new method, results obtained with this method were compared to those obtained manually by an experienced echocardiographer on Doppler echocardiographic images of left ventricular outflow tract and transvalvular flow velocity signals recorded in 30 patients, 15 with aortic stenosis and 15 with mitral stenosis. We focused on three essential variables that are measured routinely by Doppler echocardiography in the clinical setting: the maximum velocity, the mean velocity and the velocity-time integral. Comparison between the two methods has shown a very good agreement (linear correlation coefficient R(2) = 0.99 between the automatically and the manually extracted variables). Moreover, the computation time was really short, about 5s. This new method applied to DVEM could, therefore, provide a useful tool to eliminate the intra- and inter-observer variabilities associated with DVEM and thereby to improve the diagnosis of cardiovascular disease. This automatic method could also allow the echocardiographer to realize these measurements within a much shorter period of time compared to standard manual tracing method. From a practical point of view, the model developed can be easily implanted in a standard echocardiographic system.

Modifications to the basement membrane protein laminin using glycolaldehyde and A2E: a model for aging in Bruch's membrane.

In a variety of retinal diseases, including age-related macular degeneration (AMD); basement membranes are susceptible to alterations in structure and function. Chemical modifications to basement membrane proteins may deleteriously affect Bruch's membrane leading to the development of AMD. The purpose of this study was to investigate modifications from glycolaldehyde and A2E, which are present in the retinal pigment epithelium (RPE), on the membrane like protein fragment, laminin, as a model for aging of Bruch's membrane in age related eye diseases. Laminin was allowed to react with either glycolaldehyde or A2E during irradiation of A2E and then tryptically digested before analysis with electrospray ionization mass spectrometry (ESI-MS). Modifications to laminin occurred preferentially on lysine or arginine residues. The A2E modified laminin fragments are consistent with additions of A2E derived aldehydes resulting from cleavages closest to the pyridinium ring in A2E and oxidized A2E. These results provide evidence that A2E and advanced glycation endproducts (AGE) may be involved in modifications to essential basement membrane proteins leading to deleterious changes in the retinal pigment epithelium extracellular matrix (RPE-ECM) environment. These preliminary experiments are essential for the identification of these modifications in vivo.

Antioxidant properties of melanin in retinal pigment epithelial cells.

The retinal pigment epithelium (RPE) is a monolayer of highly pigmented cells lining the inner aspect of Bruch's membrane. This pigmentation is due to eumelanin and a possible antioxidant role of melanin is reported here. The photo-oxidation of A2E, a constituent of RPE lipofuscin, leads to the sequential addition of up to nine oxygen atoms and/or the addition or loss of two hydrogen atoms. These photo-oxidations were investigated in the presence and absence of either calf or human RPE melanin in A2E-laden RPE cells. It was found that calf melanin was protective against the photo-oxidation of A2E, with an inhibition of oxidation of up to 50% in the case of the addition of two oxygen atoms. Calf melanin was also protective against blue light-induced damage to RPE cells. In addition this ability appears to decrease in humans as they grow older. With aging, a melanin-lipofuscin complex called melanolipofuscin forms. It is suggested that the oxidation or photo-oxidation of A2E in vivo may contribute to the age-related deterioration of the anti-oxidant role of RPE melanin and lead to various retinal disorders, such as age-related macular degeneration.

Understanding the reasons for decline of HIV prevalence in Haiti.

OBJECTIVES: HIV sero-surveillance rounds and projection estimates suggest a decline of HIV prevalence among pregnant women and the general population in Haiti. This study aimed to evaluate the decline of HIV prevalence and understand the reasons for the decline. METHODS: Following an epidemiological analysis, three mathematical models were used to re-create the national epidemic, calculate HIV incidence, and confirm the decline of HIV prevalence. Declining trends in prevalence data were compared with observed trends in behavioural data. RESULTS: HIV progressed rapidly from initial infection to AIDS and death, with people dying twice as fast as in developed countries. With the rapid progression of the disease and the early intervention efforts in securing the blood supply, prevalence among commercial sex workers and blood donors peaked in the late 1980s followed by a decline in the mid-1990s in the general population. The observed decline among pregnant women and in the general population was confirmed after controlling for confounding variables. The Haitians are well informed: there is an increase in condom use with occasional partners at last contact and in abstinence and fidelity, and a decrease in the number of occasional partners. However, the age of sexual debut is lower and the proportion of sexually active youth has increased. CONCLUSIONS: There is evidence of decline in HIV prevalence among pregnant women, specifically among pregnant women living in urban areas and pregnant women 25 years and older, but not among pregnant women living in rural areas and pregnant women 24 years and younger. Although many factors have acted in synergy to halt the AIDS epidemic in Haiti, the main reasons for decline seem to point to mortality and blood safety intervention efforts in the early stages of the epidemic.

A new approach to measuring the action spectrum for singlet oxygen production by human retinal lipofuscin.

The human retinal pigment epithelial (RPE) layer contains a complex mixture of components called lipofuscin; this mixture forms with age and with various genetic disorders such as Stargardt's disease. Its presence may contribute to retinal deterioration via several mechanisms including photochemical processes. In the lipofuscin mixture, both type I and II mechanisms have been identified, with the latter consisting of the generation of singlet oxygen. Several components of that mixture have been identified, most notably a bis-retinoid pyridinium compound called A2E and its derivatives. Photooxidative studies on the compound A2E have revealed that its dominant photochemical mechanism is via free radical or type I processes. Because singlet oxygen is an important photooxidative intermediate in tissue, its generation in the RPE may contribute to retinal maculopathies. It is therefore necessary to determine which specific component(s) in the lipofuscin mixture produce singlet oxygen upon excitation with light. This was ascertained by evaluating the action spectrum for singlet oxygen production for the whole lipofuscin mixture using time-resolved spectroscopy. Singlet oxygen was generated by excitation of the sample at different wavelengths while maintaining a constant beam energy, and was directly detected by its phosphorescence decay at 1270 nm using a Ge photodiode. The action spectrum for singlet oxygen sensitization by the organic soluble portion of lipofuscin had an absorption maximum at ca 380 nm, which is to the blue of A2E (maximum at 430 nm). Compounds with a similar absorption maximum eluted in the HPLC earlier than A2E and were detected in human lipofuscin. The concentration of this component apparently increased in concentration in human RPE lipofuscin mixture as a function of age up to 90 years old.

Nasal potential difference increases with gestation in moderately preterm neonates on the first postnatal day.

At birth the mammalian airway switches from liquid secretion to absorption, an important mechanism in lung liquid clearance. Airway ion transport was examined on the first postnatal day in 38 moderately preterm infants (29-36 weeks gestation). The absorptive airway ion transport capacity was well developed regardless of respiratory condition and there was little capacity for Cl- secretion.

In vivo measurement of time-resolved autofluorescence at the human fundus.

An experimental setup for measurement of time-resolved autofluorescence of the human eye fundus is demonstrated. The method combines laser scanning technique and time-correlated single photon counting. The light source is a laser diode, delivering pulses of about 100 ps duration at a repetition rate of 40 MHz. The excitation wavelength is 446 nm and the cutoff wavelength of fluorescence detection is at 475 nm. The autofluorescence can be determined with a spatial resolution of 80 x 80 microm2 and 25 ps time resolution. The fluorescence decay is optimally approximated by a biexponential model. The dominating lifetime tau1 is shortest in the macula (320 to 380 ps) and reaches 1500 ps in the optic disk. The lifetime tau2 varies between 2 ns and 5 ns, but the spatial distribution is more homogeneous. Respiration of 100% oxygen for 6 min leads to changes in the fluorescence lifetime pointing to detection of coenzymes. Diagrams of lifetime tau2 versus tau1 are well suited for comparison of substances. Such lifetime clusters of a 20 deg macular field of a young healthy subject and of a patient suffering from dry age-related macular degeneration overlap only partially with tau2-tau1 clusters of lipofuscin.