Optimisation of growth conditions for ovine airway epithelial cell differentiation at an air-liquid interface

Respiratory tract infections are of significant concern in the agriculture industry. There is a requirement for the development of well-characterised in vitro epithelial cell culture models in order to dissect the diverse molecular interactions occurring at the host-pathogen interface in airway epithelia. We have analysed key factors that influence growth and differentiation of ovine tracheal epithelial cells in an air-liquid interface (ALI) culture system. Cellular differentiation was assessed at 21 days post-ALI, a time-point which we have previously shown to be sufficient for differentiation in standard growth conditions. We identified a dose-dependent response to epidermal growth factor (EGF) in terms of both epithelial thickening and ciliation levels. Maximal ciliation levels were observed with 25 ng ml-1 EGF. We identified a strict requirement for retinoic acid (RA) in epithelial differentiation as RA exclusion resulted in the formation of a stratified squamous epithelium, devoid of cilia. The pore-density of the growth substrate also had an influence on differentiation as high pore-density inserts yielded higher levels of ciliation and more uniform cell layers than low pore-density inserts. Differentiation was also improved by culturing the cells in an atmosphere of sub-ambient oxygen concentration. We compared two submerged growth media and observed differences in the rate of proliferation/expansion, barrier formation and also in terminal differentiation. Taken together, these results indicate important differences between the response of ovine tracheal epithelial cells and other previously described airway epithelial models, to a variety of environmental conditions. These data also indicate that the phenotype of ovine tracheal epithelial cells can be tailored in vitro by precise modulation of growth conditions, thereby yielding a customisable, potential infection model

Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface

The respiratory tract and lungs are subject to diverse pathologies with wide-ranging implications for both human and animal welfare. The development and detailed characterization of cell culture models for studying such forms of disease is of critical importance. In recent years the use of air-liquid interface (ALI)-cultured airway epithelial cells has increased markedly, as this method of culture results in the formation of a highly representative, organotypic in vitro model system. In this study we have expanded on previous knowledge of differentiated ovine tracheal epithelial cells by analysing the progression of differentiation over an extensive time course at an ALI. We observed a pseudo-stratified epithelium with ciliation and a concurrent increase in cell layer thickness from 9 days post-ALI with ciliation approaching a maximum level at day 24. A similar pattern was observed with respect to mucus production with intensely stained PAS-positive cells appearing at day 12. Ultrastructural analysis by SEM confirmed the presence of both ciliated cells and mucus globules on the epithelial surface within this time-frame. Trans-epithelial electrical resistance (TEER) peaked at 1049 Ω × cm2 as the cell layer became confluent, followed by a subsequent reduction as differentiation proceeded and stabilization at ~200 Ω × cm2. Importantly, little deterioration or de-differentiation was observed over the 45 day time-course indicating that the model is suitable for long-term experiments

Differentiated ovine tracheal epithelial cells support the colonisation of pathogenic and non-pathogenic strains of Mannheimia haemolytica

Mannheimia haemolytica is the primary bacterial species associated with respiratory disease of ruminants. A lack of cost-effective, reproducible models for the study of M. haemolytica pathogenesis has hampered efforts to better understand the molecular interactions governing disease progression. We employed a highly optimised ovine tracheal epithelial cell model to assess the colonisation of various pathogenic and non-pathogenic M. haemolytica isolates of bovine and ovine origin. Comparison of single representative pathogenic and non-pathogenic ovine isolates over ten time-points by enumeration of tissue-associated bacteria, histology, immunofluorescence microscopy and scanning electron microscopy revealed temporal differences in adhesion, proliferation, bacterial cell physiology and host cell responses. Comparison of eight isolates of bovine and ovine origin at three key time-points (2 h, 48 h and 72 h), revealed that colonisation was not strictly pathogen or serotype specific, with isolates of serotype A1, A2, A6 and A12 being capable of colonising the cell layer regardless of host species or disease status of the host. A trend towards increased proliferative capacity by pathogenic ovine isolates was observed. These results indicate that the host-specific nature of M. haemolytica infection may result at least partially from the colonisation-related processes of adhesion, invasion and proliferation at the epithelial interface

Assessing the quality of life of the individual: the SEIQoL with a healthy and a gastroenterology unit population

Current methods of measuring quality of life (QoL) impose an external value system on individuals, rather than allowing them to describe their lives in terms of those factors which they consider important. The Schedule for the Evaluation of Individual Quality of Life (SEIQoL) was developed to overcome such limitations. The QoL of 42 healthy attenders at an international immunization clinic was assessed using SEIQoL. Judgement reliability was high (r = 0·74) and individuals' judgement policies accounted for a large percentage of the variance in overall QoL (R2 = 0·75) demonstrating the construct validity of judgement analysis in this context. In a second study of QoL of out-patients suffering from irritable bowel syndrome (IBS) (N = 20) or peptic ulcer disease (PUD) (N = 20) was assessed using SEIQoL. Judgement reliability was lower (r = 0·54) although statistically highly significant (P < 0·01), and the variance in overall QoL judgements explained was high (R2 = 0·74). SEIQoL is an acceptable, reliable and valid technique for measuring individual QoL that takes greater account of individual perspectives than traditional measurement approache

The role of psychological flexibility in appearance anxiety in people who have experienced a visible burn injury

Background Individuals with visible differences can experience appearance anxiety that is distressing and disruptive to daily functioning. Understanding psychological factors that maintain appearance anxiety related to scarring is important in developing theoretical understanding of adjustment to injury, and in identifying targets for psychological therapies. This study aimed to investigate whether psychological flexibility, a key element underpinning acceptance and commitment therapy (ACT), was associated with appearance anxiety. It was hypothesised that reduced psychological flexibility (lower acceptance, cognitive defusion, mindfulness, and committed action) would be related to increased appearance anxiety. The role of psychological flexibility in the maintenance of appearance anxiety was investigated using a cross-sectional quantitative questionnaire study. Method Seventy-eight burns patients (47 female, 31 male; M age = 45.2 years) completed the Derriford Appearance Scale (DAS-24), the Acceptance and Action Questionnaire (AAQ-II), the Cognitive Fusion Questionnaire (CFQ), the Five Facet Mindfulness Questionnaire (FFMQ), and the Committed Action Questionnaire (CAQ-8). Results As hypothesised, increased appearance anxiety was related to reduced acceptance (rs(76) = 0.80, p < 0.001, one-tailed) and cognitive defusion (rs(76) = 0.76, p < 0.001). Reduced levels of mindfully describing (r(72) = −0.39, p < 0.001), acting with awareness (r(72) = −0.57, p < 0.001), non-judging (r(72) = −0.61, p < 0.001) and non-reactivity (r(72) = −0.28, p < 0.01) as well as reduced committed action (r(72) = −0.57, p < 0.001) were also related to increased appearance anxiety. Conclusions Individuals experiencing appearance anxiety associated with a burn injury may struggle with accepting difficult emotions, stepping back from distressing thoughts, being mindful and engaging in valued action. These findings suggest that ACT may be useful in treating appearance related anxiety and concerns associated with conditions causing a visible difference

Are there valid proxy measures of clinical behaviour?

Background: Accurate measures of health professionals' clinical practice are critically important to guide health policy decisions, as well as for professional self-evaluation and for research-based investigation of clinical practice and process of care. It is often not feasible or ethical to measure behaviour through direct observation, and rigorous behavioural measures are difficult and costly to use. The aim of this review was to identify the current evidence relating to the relationships between proxy measures and direct measures of clinical behaviour. In particular, the accuracy of medical record review, clinician self-reported and patient-reported behaviour was assessed relative to directly observed behaviour. Methods: We searched: PsycINFO; MEDLINE; EMBASE; CINAHL; Cochrane Central Register of Controlled Trials; science/social science citation index; Current contents (social & behavioural med/clinical med); ISI conference proceedings; and Index to Theses. Inclusion criteria: empirical, quantitative studies; and examining clinical behaviours. An independent, direct measure of behaviour (by standardised patient, other trained observer or by video/audio recording) was considered the 'gold standard' for comparison. Proxy measures of behaviour included: retrospective self-report; patient-report; or chart-review. All titles, abstracts, and full text articles retrieved by electronic searching were screened for inclusion and abstracted independently by two reviewers. Disagreements were resolved by discussion with a third reviewer where necessary. Results: Fifteen reports originating from 11 studies met the inclusion criteria. The method of direct measurement was by standardised patient in six reports, trained observer in three reports, and audio/video recording in six reports. Multiple proxy measures of behaviour were compared in five of 15 reports. Only four of 15 reports used appropriate statistical methods to compare measures. Some direct measures failed to meet our validity criteria. The accuracy of patient report and chart review as proxy measures varied considerably across a wide range of clinical actions. The evidence for clinician self-report was inconclusive. Conclusion: Valid measures of clinical behaviour are of fundamental importance to accurately identify gaps in care delivery, improve quality of care, and ultimately to improve patient care. However, the evidence base for three commonly used proxy measures of clinicians' behaviour is very limited. Further research is needed to better establish the methods of development, application, and analysis for a range of both direct and proxy measures of behaviour

How Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) Progresses: The Natural History of ME/CFS.

We propose a framework for understanding and interpreting the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) that considers wider determinants of health and long-term temporal variation in pathophysiological features and disease phenotype throughout the natural history of the disease. As in other chronic diseases, ME/CFS evolves through different stages, from asymptomatic predisposition, progressing to a prodromal stage, and then to symptomatic disease. Disease incidence depends on genetic makeup and environment factors, the exposure to singular or repeated insults, and the nature of the host response. In people who develop ME/CFS, normal homeostatic processes in response to adverse insults may be replaced by aberrant responses leading to dysfunctional states. Thus, the predominantly neuro-immune manifestations, underlined by a hyper-metabolic state, that characterize early disease, may be followed by various processes leading to multi-systemic abnormalities and related symptoms. This abnormal state and the effects of a range of mediators such as products of oxidative and nitrosamine stress, may lead to progressive cell and metabolic dysfunction culminating in a hypometabolic state with low energy production. These processes do not seem to happen uniformly; although a spiraling of progressive inter-related and self-sustaining abnormalities may ensue, reversion to states of milder abnormalities is possible if the host is able to restate responses to improve homeostatic equilibrium. With time variation in disease presentation, no single ME/CFS case description, set of diagnostic criteria, or molecular feature is currently representative of all patients at different disease stages. While acknowledging its limitations due to the incomplete research evidence, we suggest the proposed framework may support future research design and health care interventions for people with ME/CFS