Thesis (doctoral)--Freie Universität Berlin, 1979.

Background: Survival for people with lung cancer is poor and inequalities in access to care have been demonstrated. Methods: Primary care data from The Health Improvement Network, secondary care data from Hospital Episodes Statistics and the National Lung Cancer Audit, and death records from the Office for National Statistics, were used to investigate clinical questions in lung cancer. Matched case-control methodology was used to investigate the association between sex, smoking quantity, chronic obstructive lung disease (COPD), and lung cancer. Case control and cohort studies were performed to investigate early mortality after lung cancer surgery and treatment decisions in small cell lung cancer (SCLC). Multivariate logistic regression was used to generate a predictive score for early mortality after lung cancer surgery. Results: Sex significantly modified the effect of smoking on lung cancer with women at higher risk for the same quantity smoked. COPD was strongly associated with lung cancer in univariate analysis however this was heavily confounded by smoking and strongly related to timing of diagnosis. For people with non-small cell lung cancer, 90-day mortality after surgery was 5.9%. Age, co-morbidity index, performance status, procedure type and stage were significantly associated with this outcome and therefore make up the predictive score. 70% of people with SCLC were treated with chemotherapy however this varied according to several factors including referral method and socioeconomic status. Survival after chemotherapy for people with SCLC was similar to that reported in clinical trials. Conclusions: The work in thesis provides further evidence that women are at higher risk of lung cancer per quantity of cigarettes smoked, and challenges the commonly held belief that COPD is a strong independent risk factor for lung cancer. In addition it produced a predictive score for early mortality following lung cancer surgery, and provides information on treatment decisions and outcomes for SCLC.

As the first line of defence against external stimuli, the airway epithelium undergoes frequent injury during an adult life. This is countered by repair mechanisms that ensure the integrity of the epithelium. It has been established that there are resident stem/progenitor cells localized within specific niches throughout the respiratory tract. Stem/progenitor cells are activated according to the severity of the insult and are thought to be responsible for repairing the airway epithelium. It has been difficult to isolate those stem cells. In this study, an in vitro human bronchial epithelial model was adapted and characterised. In culture, a pseudostratified epithelium was observed, with basal, ciliated, and secretory cells. Mucus production was also seen in this model. A scrape-wound of the model was employed to study the responses of the airway epithelium to injury. It was observed that migration and then proliferation, of CD44 expressing basal cells, are the first events that take place after scrape-wounding. Up-regulation of CD44 was also observed at the edge of the wounds early post-wounding. This suggested a key role for CD44-expressing basal cells in migration and proliferation after wounding, also suggesting this population may contain a progenitor cell population. Investigation of the secretory profile of the airway epithelium post-wounding revealed an increase in a number of cytokines and growth factors. In particular, IL-6, IL-8, ENA-78, and RANTES were all elevated compared with unwounded cultures. A side population (SP) was identified in differentiated and undifferentiated human bronchial epithelial cells in at least some cultures accounting for 0.1-1.15% of cells present. In summary, the epithelium is important in airway wound repair with basal cells appearing to contain the progenitor population in this human bronchial epithelial cell (HBEC) model. Moreover, SP studies suggested the presence of SP in at least some cultures, which might contribute to airway regeneration. The secretory profile of the airway epithelium post-wounding indicates up-regulation of specific cytokines, which may be important in the pathogenesis of lung diseases such as asthma and COPD. This model should prove useful to assess wound repair pathways and may be of use in the future for proof of concept studies on novel therapeutic agents.

Childhood asthma is an extensive problem and is particularly pronounced in the UK. Asthma can restrict activities, cause school absence and can be the source of considerable stress in both children and their parents. Mortality is rare and preventable, although poor perception of symptoms and delay in seeking medical attention are strong risk factors for a fatal asthma attack. Self- management actively involves the child in their own healthcare and entails monitoring symptoms and responding accordingly and has been linked to better outcomes. This can be facilitated by health education. The aims of this project were to develop an educational computer program to promote self-management skills in children and young people with asthma, to evaluate its effectiveness in a clinical sample and to validate measures of asthma knowledge and locus of control. The Asthma Files uses a 'secret agent' theme to encourage users to investigate information about asthma. The program was piloted with 28 children aged 7-16 over a one year period and revised in accordance with both qualitative and quantitative data obtained. To evaluate the computer program, 101 children aged between 7 and 14 years were recruited from three hospital asthma out-patient clinics to participate in a randomised, controlled trial. They were interviewed using asthma knowledge and asthma-specific locus of control measures developed and validated for the purposes of the study. All children were given an information booklet one month later and, in addition, 50 children used the computer program. Baseline knowledge levels were low. At one-month follow-up (n=99), children in the computer group had significantly greater increases than those in the control group (p<0.001), along with an rise in internal locus of control(p<0.01). There was no evidence of changes in objective lung function measures, hospitalisations or oral steroid use between the groups at this time. However, at six months follow-up (n=90), children in the computer group were significantly less likely to have required oral steroids or school absence than the control group (p<0.05). The program was popular with the children across the age range and received positive feedback on both content and mode of delivery. Responding to comments provided by the children in the RCT, some minor amendments were made to the program, which is now available for public use. The Asthma Files computer program was successful in increasing knowledge and promoting internal locus of control. More research is needed to evaluated how this might translate into longer term improvements in self-management. NB. This ethesis has been created by scanning the typescript original and may contain inaccuracies. In case of difficulty, please refer to the original text.

Asthma is an inflammatory disease of the airways characterised by airway remodelling and hyperresponsiveness. New treatments are needed for patients with severe asthma whose disease is not controlled with currently available therapies. Asthma pathophysiology is complex, however, accumulating evidence suggests multiple inflammatory pathways in asthma converge onto a relatively small number of downstream targets that may be of therapeutic interest. These include mitogen activated protein kinases (MAPKs), the pro-inflammatory transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) and transcriptional regulators such as histone acetyl transferases (HATs) and histone deacetylases (HDACs). Chemokines are molecules secreted at sties of inflammation, attracting inflammatory cells and perpetuating the inflammatory response. Here we studied the mechanisms by which the pro-inflammatory mediator endothelin-1 (ET-1) and the cytokine tumour necrosis factor-alpha (TNF-alpha) promoted expression by primary human airway smooth muscle cells (HASMC) of two important chemokines, monocyte chemotactic protein-1 (MCP-1) and eotaxin. Further, we studied the mechanisms by which existing asthma therapies (long acting beta agonists (LABA) and glucocorticoids) modulated TNF-alpha-stimulated eotaxin expression. Endothelin-1 stimulated MCP-1 release through a transcriptional mechanism involving NF-kappaB and AP-1; the upstream signalling pathway involved p38 and p44/p42 MAPKs. Previously, this lab showed that TNF-alpha-induced eotaxin release is also NF-kappaB-dependent, involving histone H4 acetylation at the eotaxin promoter. Here we found that TNF-alpha-induced eotaxin release does not involve histone H3 acetylation, and that TNF-alpha-dependent histone H4 acetylation does not occur through alterations in total histone activity or levels of the key HDACs -1 and -2. Similarly, modulation of TNF-alpha effects on eotaxin expression by glucocorticoids and LABA is independent of total HDAC activity and HDAC-1 and -2 levels. These studies support the body of evidence suggesting that multiple inflammatory pathways in asthma converge onto a small number of downstream targets, and are relevant to the understanding and treatment of asthma.

Asthma is a chronic inflammatory disease of the airways characterised by airway hyper-responsiveness (AHR), inflammation of the airways and reversible airway obstruction. Airway remodelling is a feature of asthma, especially in cases of severe and fatal asthma, and includes structural changes such as increased airway smooth muscle (ASM) mass, mucous gland hyperplasia, subepithelial fibrosis and angiogenesis. TGF-β is a pleiotropic cytokine that has been implicated in the development of many of these changes. However, TGF-β is released from cells in a latent complex, associated with its pro-peptide the latency associated peptide (LAP). Extracellular activation of latent TGF-β is the rate limiting step in TGF-β bioavailability. Although TGF-β activation has been investigated in airway epithelial cells, to date, no studies have investigated TGF-β activation by airway smooth muscle cells. The hypothesis of this thesis is therefore that human airway smooth muscle cells can activate TGF-β in vitro. The hypothesis of this thesis has been tested by investigating effects of the serine protease mast cell tryptase, mechanical wounding of cell mono layers and the phospholipid lysophosphatidic acid (LPA) on TGF-β activation by primary airway smooth muscle cells in vitro. We have utilised transformed mink lung epithelial cells, a reporter cell that express a TGF-β responsive promoter driving a luciferase gene, and quantitative PCR for the TGF-β-inducible gene plasminogen activator inhibitor-1 (PAI1) to investigate TGF-β activation. Moreover, we show for the first time that TGF-β activation can be assessed in vitro by detecting the translocation of Smad 2 and 3 from the cytoplasm to the nucleus by western blotting. The results presented in this thesis provide evidence that airway smooth muscle cells are capable of activating TGF-β in vitro. These data show that the serine protease tryptase, released from activated mast cells, can proteolytically activate TGF-β via a mechanism that is independent of the tryptase receptor protease activated receptor-2 (PAR2). This effect is not accompanied by increased expression of the latent TGF-β complex. Furthermore, these data provide evidence that airway smooth muscle cells can activate TGF-β via the integrin αβV5 in response to LPA stimulation. We have found that cells from asthmatic patients activate more TGF-β in response to LPA than cells from non-asthmatic individuals and this is not due to a difference in cell surface expression levels of the αβV5 integrin. LPA-induced TGF-β activation can be inhibited by the β2 adrenoreceptor agonist formoterol, which is a commonly used asthma therapy, and the muscarinic receptor agonist methacholine, which causes cell contraction, also causes TGF-β activation by airway smooth muscle cells. Furthermore, the data presented here show that the cytoplasmic domain of the integrin β5 subunit interacts with the cytoskeletal protein talin to mediate TGF-β activation. Together, these data highlight two previously unreported, biologically relevant, mechanisms of TGF-β activation employed by airway smooth muscle cells in vitro, both of which could contribute to the development of airway remodelling in asthma in vivo. Data concerning a αβV5 mediated TGF-β activation has led us to hypothesise that contraction of airway smooth muscle leads to TGF-β activation in vivo. If correct, this could be vital to our understanding of how airway remodelling is initiated in asthma, and could lead to the development of new therapies aimed at inhibiting contraction-induced TGF-β activation, for the treatment of asthma.

Chronic Obstructive Pulmonary Disease (COPD) presents an enormous public health challenge. Cigarette smoking remains the most important aetiological factor and although legislation to reduce smoking has been introduced in parts of the more developed world, consumption is increasing in many of the poorest parts of the world. With the predicted rise in disease prevalence, COPD is expected to become the worlds third largest cause of death by 2020. COPD is a disease state characterised by airflow limitation that is not fully reversible. Inhaled bronchodilators can only produce a small improvement in the airflow obstruction, but despite this, patients with COPD frequently use high doses of beta-2-agonists as the disease progresses and they develop breathlessness and exercise limitation. Short-acting beta-2-agonists are generally used as required to reduce breathlessness and reduce airflow obstruction whereas long-acting beta-2-agonists are prescribed on a regular twice-daily basis to reduce symptoms and rescue medication use and because of a potential beneficial effect on quality of life and exacerbation rates. Although generally well tolerated, the safety of inhaled beta-2-agonists has been a source of some concern since the late 1960s, when an epidemic of asthma deaths was associated with the use of a high dose formulation of isoprenaline. Further controversy has followed and questions have extended to long-acting beta-2-agonists, most notably after a recent large-scale post marketing surveillance study identified an association between the regular use of inhaled salmeterol and asthma-related deaths. The safety of inhaled beta-2-agonists is also an important consideration for patients with COPD. Being older and likely to have a history of cigarette consumption means that they are at risk of having symptomatic, or subclinical, ischaemic heart disease. Beta-2-agonists cause a number of systemic effects including an increase in heart rate, transient hypoxaemia and hypokalaemia. Since many patients with COPD are already hypoxaemic and may be taking other drugs that stimulate the myocardium and cause hypokalaemia, the additional systemic effects from beta-2-agonists may be more likely to produce adverse cardiac events including dysrhythmia and ischaemia. This thesis is concerned with the safety of inhaled beta-2-agonists in the management of COPD. The introduction consists of an overview of the epidemiology, natural history and pathology of COPD (Chapter 1) and a review of human beta-2-adrenoceptor function and inhaled beta-2-agonist pharmacology (Chapter 2). This is followed by a systematic literature review of the results from long-term clinical studies of inhaled beta-2-agonists in subjects with COPD (Chapter 3). The original work consists of three clinical studies that have examined aspects of the effect of high dose inhaled beta-2-agonists in subjects with COPD and a discussion to place these findings in context. Most published studies of inhaled beta-2-agonists in subjects with COPD have focused on their efficacy, rather than safety. We were concerned that some individuals with COPD and limited bronchodilator reversibility may experience an increase in adverse systemic effects after inhaling high doses of beta-2-agonists, which could lead to detrimental outcomes in certain clinical situations. Apart from the cardiac effects mentioned above, beta-2-agonists increase tremor, which causes CO2 production, and cardiac output and tissue perfuson, which increases the transport of CO2 to the lungs. The increase in CO2 flux to the lungs will normally increase ventilation. We were concerned however that some subjects with severe COPD would not be able to increase ventilation appropriately in response to the beta-2-agonist and this would lead to an increase in PaCO2. Our hypothesis was that high dose inhaled beta-2-agonists could worsen respiratory failure in some subjects with severe COPD. The first two studies in the thesis examined the effect of high dose inhaled salbutamol on the partial pressure of arterial oxygen and carbon dioxide in subjects with severe COPD. We initially conducted a double blind, randomised study on subjects within 48 hours of being admitted to hospital with an acute exacerbation of COPD (Chapter 4). The study was designed to determine whether high dose salbutamol caused an increase in the partial pressure of arterial carbon dioxide. We randomised subjects at a ratio of 3:1 to receive either salbutamol or ipratropium bromide and studied the pharmacodynamic effect on heart rate, PaO2 and PaCO2 over five hours. Over eighteen months and despite extensive efforts I was only able to recruit ten subjects, of whom five completed the study. I found that subjects who required hospital admission with an acute exacerbation of COPD were either too unwell for the study, had co-morbidities that precluded participation or the individuals were unwilling to participate. Although the study was terminated prematurely and we were unable to perform statistical analysis, I have presented the findings from the five subjects who completed the study, of whom four were randomised to receive salbutamol. We used ascending doses of salbutamol (1.25mg, 1.25mg, 25mg, 5mg, 5mg) and found no consistent effect on PaCO2 or PaO2 and no dose response relationship. The subject with the highest baseline PaCO2 did however have a rise in PaCO2 with the highest 5mg doses of salbutamol. To test the hypothesis further we conducted a randomised, double blind, crossover study and examined the effect of salbutamol on the arterial blood gas tensions of fourteen patients with stable severe COPD and a history of chronic or intermittent hypercapnia. The study was designed to determine whether high dose salbutamol causes a rise in PaCO2 when inhaled by subjects with severe COPD and a history of alveolar hypoventilation. We compared the effect of two 5mg doses with two 200 microgram doses of salbutamol on PaO2 and PaCO2 and heart rate. The subjects had severe COPD with a mean FEV1 of 0.71 L (27% predicted) and a mean smoking history of 53 pack years. The mean baseline PaO2 was 7.9 kPa and the mean baseline PaCO2 was 7.0 kPa. The high dose of salbutamol caused a mean fall in both PaO2 and PaCO2 and a small increase in heart rate. There was some support for our hypothesis however as three subjects had a small rise in PaCO2 after high dose nebulised salbutamol (Chapter 5). The third study was a double blind, crossover, dose-response examination of the bronchodilator and systemic effects of inhaled formoterol in subjects with COPD (Chapter 6). The rapid onset and prolonged duration of action of formoterol offers potential for the drug to be used as rescue medication in addition to twice daily maintenance therapy, as is the case in the management of asthma. Our hypothesis was that high doses of formoterol would produce adverse systemic effects that would outweigh the beneficial bronchodilator effects in subjects with COPD and limited bronchodilator response to salbutamol. We studied 20 subjects, with a mean FEV1 of 1.32 L (47% predicted) and a mean smoking history of 42 pack years. Each subject was studied on five days and after receiving placebo, formoterol 6, 12, 24 and 48 mg in a random sequence, we examined the effect of each dose on FEV1, tremor, dyspnoea, heart rate, blood pressure, SpO2, walk distance, potassium and satisfaction. We found that all doses were well tolerated and although there was a small dose related increase in FEV1 and the mean satisfaction scores were higher with each dose of formoterol than placebo, there was no dose related improvement in measures that are important to the patient, including breathlessness and walk distance. Apart from a dose related increase in tremor, other systemic effects were limited. All three studies found that high dose inhaled beta-2-agonists produced relatively modest systemic effects in subjects with COPD.

This thesis used a qualititave research approach to explore the causes, manifestations and consequences of tuberculosis (TB) stigma in an urban district in Ghana. It examined reasons why TB is stigmatised and elucidated how TB stigma manifests within the community setting and the healthcare system. It also explored the feelings and experiences of TB patients, to highlight how the fear of stigmatisation may affect case finding and treatment adherence. Twenty eight focus groups (6 with patients, 6 with health workers and 16 with community members) and 121 individual interviews (66 with community members, 34 with patients and 21 with health staff) were conducted. Data were analysed using Grounded Theory techniques and procedures. Eleven causes of TB stigma were identified: fear of infection; physical frailty of TB patients; association of TB with HIV/AIDS; perceived causes and spread of TB; outdated societal practices about TB; public health practice and discourse; attitudes of healthcare workers towards TB patients; health staff’s own fear of TB; self-stigmatisation by TB patients; judgement, blaming and shaming TB patients; and past experiences with TB. Elements of physical and moral threats were identified in all these causes of TB stigma. The threat the disease poses to community members led to imposition of socio-physical distance, participatory restrictions and rules for unexpected interactions on those suffering from TB in society. Within the healthcare system, the threat of TB affected the attitudes and behaviours of healthcare workers towards TB patients and TB work. Health managers also sited TB units/wards in isolated parts of the hospital, and failed to provide adequate tools and equipment, support and supervision to enable the provision of quality TB services. The fear of stigmatisation made the patients deny the obvious symptoms of the disease, and report to the hospital only after prolonged period of self-medication in the community. When diagnosed, they cried, questioned how they got the disease, contemplated committing suicide and were mostly isolated within the family and community. For everyone, the threat of TB underlies their beliefs, attitudes, actions and behaviours when interacting with TB patients. It also forms the basis of avoidance of social interactions, and attitudes and practices of healthcare workers towards TB patients. The TB control programme should encourage open discussion about TB in the community and tailor health education messages to the community’s understanding of the disease. TB services should be completely integrated into the general healthcare system and community members involved in activities of the TB control programme. Regular refresher courses in TB control and management should be organised for health professionals and a national guideline for the prevention of TB in health workers developed.

The Single combination budesonide/formoterol inhaler as Maintenance And Reliever Therapy (SMART) regimen reduces severe asthma exacerbations, but it is uncertain whether it increases the risk of adverse effects due to high corticosteroid and beta-agonist doses with both short-term and cumulative exposure in patients at risk of severe exacerbations. The primary hypothesis was that the SMART regimen would reduce the risk of beta-agonist overuse. Secondary aims were to investigate whether patients treated with the SMART regimen were less likely to seek medical review in the setting of beta-agonist overuse and to determine whether any reduction in severe asthma exacerbations would be at a cost of a higher systemic corticosteroid burden. This 24-week, open-label, parallel-group, multicentre randomised controlled trial randomised 303 asthma patients with a recent exacerbation to combination 200/6µg budesonide/formoterol metered dose inhaler (MDI) according to the SMART regimen (two actuations twice daily as maintenance with one extra actuation as-needed for relief of symptoms) or a fixed-dose regimen (two actuations twice daily as maintenance) with one to two actuations of 100µg salbutamol MDI as-needed for relief of symptoms (the ‘Standard’ regimen), with electronic monitoring to measure actual medication use. The use of electronic monitoring allowed beta-agonist overuse to be applied as a marker of the risk of life-threatening asthma. The primary outcome was the proportion of participants with at least one high beta-agonist use episode (more than eight actuations per day of budesonide/formoterol in addition to the four maintenance doses in the SMART group or more than 16 actuations per day of salbutamol in the Standard group). There was no significant difference between groups in the proportion of participants with at least one high use episode: SMART 84/151 (55.6%) versus Standard 68/152 (44.7%), relative risk (95% CI) 1.24 (0.99 to 1.56), p=0.058. There were fewer days of high use in the SMART group [mean (SD) 5.1 days (14.3) versus 8.9 days (20.9), relative rate (95% CI) 0.58 (0.39 to 0.88), p=0.01]. Of the participants who had at least one high use episode, those in the SMART group had fewer days of high use without medical review [mean (SD) 8.5 days (17.8) versus 18.3 days (24.8), relative rate (95% CI) 0.49 (0.31 to 0.75), p=0.001]. The SMART regimen resulted in higher inhaled corticosteroid exposure [mean (SD) 943.5µg budesonide per day (1502.5) versus 684.3µg budesonide per day (390.5), ratio of means (95% CI) 1.22 (1.06 to 1.41), p=0.006], but reduced oral corticosteroid exposure [mean (SD) 77.5mg prednisone (240.5) versus 126.6mg prednisone (382.1), p=0.011], with no significant difference in composite systemic corticosteroid exposure [mean (SD) 793.7mg prednisone equivalent per year (893.1) versus 772.1mg prednisone equivalent per year (1062.7), ratio of means (95% CI) 1.03 (0.86 to 1.22), p=0.76]. Participants in the SMART group had fewer severe asthma exacerbations [35 (weighted mean rate per year 0.53) versus 66 (0.97), relative rate (95% CI) 0.54 (0.36 to 0.82), p=0.004]. The SMART regimen has a favourable risk/benefit profile in patients at risk of severe asthma exacerbations. Funding This study was funded by the Health Research Council of New Zealand, a government funding organisation.

Background This thesis investigates the effect of sodium modification on asthma and heart rate variability (HRV) using a randomised controlled study design. Cross-sectional data were used to assess novel markers of asthma control and if serum osmolality was inversely associated with lung function. Methods and Results The effect of dietary sodium modification on asthma control and HRV The study design was a randomised placebo-controlled double-blind study of participants aged 18-65 years old with asthma and measurable bronchial reactivity. The intervention involved adopting a low sodium diet for six weeks. 199 participants provided measures of asthma control and 29 participants provided 24-hour electrocardiographic measurements at baseline and six weeks later. There were no differences between those allocated to the low and normal sodium diet groups in any of the measures of asthma control or HRV. Bronchoconstriction induced by deep inspiration and exhaled carbon monoxide as surrogate marker of asthma control Serial PEFR measures were provided by 127 individuals to measure bronchoconstriction after deep inhalation and 182 individuals provided measurements of eCO. The relationship between these measures and asthma control was assessed. There was no relationship between either bronchoconstriction after deep inspiration or eCO and asthma control. Serum osmolality and lung function The NHANES III database was used to investigate if serum osmolality was cross-sectionally associated with lung function. In 13,602 individuals a SD increase in serum osmolality was associated with a decrease in FEV1 of 19.8ml (95% CI; -30.3 to -9.3) and FVC of 35.3ml (95% CI; -47.9 to -22.7). Conclusions A low sodium diet did not improve asthma control or increase heart rate variability in adults with asthma. Bronchoconstriction to deep inspiration and measurements of eCO have limited potential as markers of asthma control. In the NHANES dataset, increased serum osmolality was associated with decreased lung function.