Hannah J Durrington

The circadian clock and asthma

It is characteristic of asthma that symptoms worsen overnight, particularly in the early hours of the morning. Nocturnal symptoms in asthma are common and are an important indicator for escalation of treatment. An extensive body of research has demonstrated that nocturnal symptoms of cough and dyspnea are accompanied by circadian variations in airway inflammation and physiologic variables, including airflow limitation and airways hyper-responsiveness. The molecular apparatus that underpins circadian variations, controlled by so called ‘clock’ genes, has recently been characterised. Clock genes control circadian rhythms both centrally, in the suprachiasmatic nucleus of the brain and peripherally, within every organ of the body. Here, we will discuss how clock genes regulate circadian rhythms. We will focus particularly on the peripheral lung clock and the peripheral immune clock and discuss how these might relate to both the pathogenesis and treatment of asthma.

A matter of time: study of circadian clocks and their role in inflammation

Circadian rhythms regulate changes in physiology, allowing organisms to respond to predictable environmental demands varying over a 24 h period. A growing body of evidence supports a key role for the circadian clock in the regulation of immune functions and inflammatory responses, which influence the understanding of infections and inflammatory diseases and their treatment. A variety of experimental methods have been used to assess the complex bidirectional crosstalk between the circadian clock and inflammation. In this review, we summarize the organization of the molecular clock, experimental methods used to study circadian rhythms, and both the inflammatory and immune consequences of circadian disturbance.

Diagnosis of asthma in symptomatic children based on measures of lung function: an analysis of data from a population-based birth cohort study

Summary Background Concerns have been expressed about asthma overdiagnosis. The UK National Institute of Health and Care Excellence (NICE) proposed a new diagnostic algorithm applying four lung function measures sequentially (ratio of forced expiratory volume in 1 s [FEV1] to forced vital capacity [FVC] <70%, bronchodilator reversibility ≥12%, fractional exhaled nitric oxide [FeNO] ≥35 parts per billion, and peak expiratory flow variability >20%). We aimed to assess the diagnostic value of three of the tests individually, and then test the proposed algorithm in symptomatic children. Methods We used follow-up data at age 13–16 years from the Manchester Asthma and Allergy Study, a prospective, population-based, birth cohort study. We initially present results for the whole population, then by subgroup of disease. To simulate the situation in primary care, we included participants reporting symptoms of wheeze, cough, or breathlessness in the previous 12 months and who were not on regular inhaled corticosteroids. We used an epidemiological definition of current asthma, defined as all three of physician-diagnosed asthma, current wheeze, and current use of asthma treatment, reported by parents in a validated questionnaire. We assigned children with negative answers to all three questions as non-asthmatic controls. We also measured spirometry, bronchodilator reversibility, and FeNO at follow-up; data for peak expiratory flow variability were not available. We calculated the proportion of participants with a current positive lung function test at each step of the algorithm, and recorded the number of participants that met our definition of asthma. Findings Of 1184 children born into the cohort, 772 attended follow-up at age 13–16 years between July 22, 2011, and Nov 11, 2014. Among 630 children who completed spirometry, FEV1:FVC was less than 70% in ten (2%) children, of whom only two (20%) had current asthma. Bronchodilator reversibility was positive in 54 (9%) of 624 children, of whom only 12 (22%) had current asthma. FeNO was 35 or more parts per billion in 115 (24%) of 485 children, of whom 29 (25%) had current asthma. Only four of 56 children with current asthma had positive results for all three tests (spirometry, bronchodilator reversibility, and FeNO). Conversely, 24 (43%) of the 56 children with current asthma were negative on all three tests. FEV1:fvc (p=0·0075) and FeNO (p<0·0001), but not bronchodilator reversibility (p=0·97), were independently associated with asthma in multivariable logistic regression models. Among children who reported recent symptoms, the diagnostic accuracy of the algorithm was poor. Interpretation Our findings challenge the proposed cutoff values for spirometry, the order in which the lung function tests are done, and the position of bronchodilator reversibility within the algorithm sequence. Until better evidence is available, the proposed NICE algorithm on asthma diagnosis should not be implemented in children. Funding UK Medical Research Council.

‘In a dark place, we find ourselves’: light intensity in critical care units

Intensive care units provide specialised care for critically ill patients around the clock. However, intensive care unit patients have disrupted circadian rhythms. Furthermore, disrupted circadian rhythms are associated with worse outcome. As light is the most powerful ‘re-setter’ of circadian rhythm, we measured light intensity on intensive care unit. Light intensity was low compared to daylight during the ‘day’; frequent bright light interruptions occurred over ‘night’. These findings are predicted to disrupt circadian rhythms and impair entrainment to external time. Bright lighting during daytime and black out masks at night might help maintain biological rhythms in critically ill patients and improve clinical outcomes.

Incidence of primary graft dysfunction after lung transplantation is altered by timing of allograft implantation

The importance of circadian factors in managing patients is poorly understood. We present two retrospective cohort studies showing that lungs reperfused between 4 and 8 AM have a higher incidence (OR 1.12; 95% CI 1.03 to 1.21; p=0.01) of primary graft dysfunction (PGD) in the first 72 hours after transplantation. Cooling of the donor lung, occurring during organ preservation, shifts the donor circadian clock causing desynchrony with the recipient. The clock protein REV-ERBα directly regulates PGD biomarkers explaining this circadian regulation while also allowing them to be manipulated with synthetic REV-ERB ligands.

The Role of the Body Clock in Asthma and COPD: Implication for Treatment

Asthma exhibits a marked time of day variation in symptoms, airway physiology, and airway inflammation. This is also seen in chronic obstructive pulmonary disease (COPD), but to a lesser extent. Our understanding of how physiological daily rhythms are regulated by the circadian clock is increasing, and there is growing evidence that the molecular clock is important in the pathogenesis of these two airway diseases. If time of day is important, then it follows that treatment of asthma and COPD should also be tailored to the most efficacious time of the day, a concept known as ‘chronotherapy’. There have been a number of studies to determine the optimal time of day at which to take medications for asthma and COPD. Some of these agents are already used ‘chronotherapeutically’ in practice (often at night-time). However, several studies investigating systemic and inhaled corticosteroids have consistently shown that the best time of day to take these medications for treating asthma is in the afternoon or early evening and not in the morning, when these medications are often prescribed. Future, large, randomized, placebo-controlled studies of systemic and inhaled corticosteroids in asthma and COPD are needed to inform clinical practice.

Time of Day Affects Eosinophil Biomarkers in Asthma: Implications for Diagnosis and treatment

potentiation appeared to be cytokine independent (data not shown). Endothelial amyloids are heat stable, protease resistant, and RNase and DNase insensitive (1). They exhibit certain features of prion disease (10); for example, they are self-replicating and transmissible among cells (1). Here, we provide evidence that nosocomial pneumonia induces lung endothelial production of t and Ab oligomeric species that impair neurological information processing, supporting the hypothesis that these amyloids contribute to insidious end-organ dysfunction and suggesting the need for a larger cohort trial addressing this issue. In our studies, injurious amyloids were detected in the cerebrospinal fluid of infected, but not in uninfected, patients. However, our current sample size is small, and we have not determined whether other bacteria, viruses, fungi, or inflammatory conditions, such as the systemic inflammatory response syndrome, also elicit this endotheliopathy. Our studies focus on the acute consequences of infection-induced amyloids; it remains unknown as to whether the mechanisms tested here represent a cause of progressive and persistent memory loss in ICU patients. Indeed, in studies moving forward, it will be essential to determine the infection-induced t and Ab oligomer fate within the lung, blood, and other peripheral organs, including the brain. It will also be essential to determine whether infection-induced amyloid production and biodistribution contributes to cardiovascular disease, stroke, renal dysfunction, and pulmonary dysfunction in the aftermath of critical illness. n

P206 A study to investigate the mechanisms underlying circadian rhythm in asthma

Background Time of day is critical in the pathogenesis of asthma, and has been realised for centuries. Symptoms of asthma are worse around 4 am, when airway restriction is at its highest. Many asthma treatments are taken in the morning or evening, however there is increasing data that steroids are more efficacious if taken mid-afternoon. Investigating the biological timing of asthma is crucial to better understand the pathogenesis of asthma, this may lead to the discovery of new drug targets, and the identification of dynamic biomarkers that change by time of day and would be useful in a future chronotherapeutics study. Aims • Define new biochemical pathways involved in the circadian variation in asthma • Determine a circadian biomarker in asthma Method We recruited 10 atopic, moderately severe asthmatics and 10 healthy volunteers to complete 4 study visits, including an overnight stay. Blood, induced sputum and breath were sampled at intervals throughout the day and night, and physiological measurements made. PBMCs were harvested from peripheral blood at 4 am and 4 pm and plated in 6 groups, control,+LPS/anti-CD3/anti CD28,+P38 i,+LPS/anti-CD3/anti CD28 +P381,+Dexamethasone,+LPS/anti-CD3/anti CD28 +dexamethasone for 2 hours. Conditioned medium was collected and frozen at −80, cell lysates were prepared for RNA extraction, and protein purification. Reference 14/NW/1352. Results There is a high amplitude circadian change in FEV1 in asthmatics compared to healthy controls. The nadir is at 4 am (figure 1). There was also an increase in sputum and serum eosinophils (a key effector cell in asthma) at 4 am compared to 4 pm in asthmatics (figure 1). Abstract P206 Figure 1 On-going workflow includes Transcript measurement from PBMCs using a combination of RNA-Sequencing and nanostring technology (for clock genes; inflammatory mediators; Signalling regulators; MAP kinase family members) Bioplex screen for expression and activation of additional MAPkinase components, and IkBa, from protein extracts and conditioned media Lipidomic analyses of serial, matched serum samples will allow analysis of the ceramide/sphingolipid pathway Breathomics analysis for volatile organic compounds in serial, matched breath samples. Discussion We have demonstrated a significant diurnal effect on asthma lung physiology and eosinophil profiles. Further downstream analysis of serum, breath and sputum is underway.

Light Intensity on Intensive Care Units

It is well recognised that the critical care environment is disruptive to maintenance of circadian rhythm and sleep-wake cycle. The normal rhythmic 24 h profiles of physiological parameters are altered in critical care patients [1-5]. This is especially seen in septic patients [6-8]. Mechanical ventilation, sedation, severity of illness and the ICU environment (noise) may all cause circadian disruption. However, it is now clear that light is the most powerful environmental influence on the circadian clock [9] (Figure 1).

P176 Diagnosing asthma in children using spirometry: evidence from a birth cohort study

Background NICE draft guidance for the diagnosis of childhood asthma proposes algorithms based on four tests of lung function (FEV1/FVC ratio, bronchodilator reversibility [BDR], FeNO, PEFR variability); a minimum of two tests must be positive to make a diagnosis. For FEV1/FVC ratio, the proposed cut-off for a positive test is <70%, or the lower limit of normal (LLN), which is neither defined nor widely available. In this algorithm, spirometry is the first-line investigation, and children with FEV1/FVC > 70% are not offered BDR. However, the diagnostic test accuracy for FEV1/FVC and BDR is unknown. Within the setting of a population-based birth cohort we investigated the value of FEV1/FVC and BDR in diagnosing asthma. Methods We assessed study participants at clinical follow-up at age 16 years using validated questionnaires and lung function measurement. Spirometry was measured according to ATS/ERS guidelines. Using the Asthma UK reference equations, we calculated LLN for FEV1/FVC. BDR was considered positive if FEV1 increased by >12% following administration of 400 mg of salbutamol. Current asthma was defined as all three of: (1) doctor-diagnosed asthma ever, (2) wheezing in the previous 12 months and (3) current use of asthma treatment. We assigned children negative to all three features as a non-asthmatic control group. Results Spirometry was available for 630 children (325 boys, age range 13.1–16.9 years), of whom 74 (11.7%) had current asthma and 403 were assigned as non-asthmatic controls. FEV1/FVC was significantly lower among asthmatics (84.1% vs. 89.2%, p < 0.001, Figure 1). Ten children (1.6%) had FEV1/FVC <70% (two in asthma group). Discriminative ability of FEV1/FVC < 70% was poor (Receiver operating characteristic curve, AUC = 0.70; sensitivity = 2.7% [2/74], specificity = 98.8% [398/403]). For the calculated FEV1/FVC LLN (74.8% for boys, 78.2% for girls), 28 children (4.4%) had FEV1/FVC<LLN (11 in asthma group). Discriminative ability of FEV1/FVC<LLN was poor (sensitivity 14.9% [11/74]; specificity 97.0% [391/403]). BDR was positive in 54 children (8.7%), of whom 12 had asthma. Discriminative ability of BDR was poor (AUC = 0.64, sensitivity = 16.2% [12/74], specificity = 93.5% [373/399]). Combining these two tests did not result in a better diagnostic accuracy (sensitivity = 2.7%, specificity = 99.0%). Abstract P176 Figure 1 Conclusions FEV1/FVC < 70% or <LLN, and BDR > 12% have a poor diagnostic accuracy as tests for childhood asthma.