Measuring the level of exhaled NO (eNO) in the breath is a new method to monitor airway inflammation in asthma and may have a role in the management of asthma. The hypotheses were that eNO will reflect the degree of inflammation in chronic asthma, and will indicate how anti- inflammatory therapy should be altered to improve asthma control. Three studies were performed to test the hypotheses. A cross sectional study was performed to define the normal range of eNO and to compare this range with those who have asthma or atopy. The second study was observational, to compare the level of eNO during and after an exacerbation of asthma. The third study was an interventional study to evaluate eNO in management of paediatric asthma. In this latter study the level of eNO was measured to monitor airway inflammation in asthmatic children with the intention of adjusting antiinflammatory drugs (inhaled glucocorticosteroids) according to the level of eNO. These studies have shown that the mean level of eNO was significantly higher in asthmatic compared with normal subjects, but not significantly different when compared with atopic non-asthmatic subjects. eNO was correlated with the number of positive skin prick tests in atopic subjects whether asthmatic or nonasthmatic. The eNO level was increased during acute exacerbations of asthma and decreased after two weeks with therapy of GCS. In a pilot study eNO appeared to be superior to FEV1 in adjusting the dose of iGCS to control asthmatic children, but this needs to be confirmed with a larger sample size. Another non-invasive method to detect inflammatory markers is the technique of exhaled breath condensate (EBC). Although NO is degraded to NOx, it was found that eNO had no significant correlation with EBC NOx but had a significant correlation with pH. Hypertonic saline challenge, an artificial model of an asthmatic exacerbation was associated with an increase in EBC volume and the release of histamine, implicating mast cell activation. These novel findings suggest that non-invasive markers can be used both for clinical and mechanistic proposes.

Doctor of Philosophy (PhD) / Asthma is an inflammatory disease characterised by tissue remodelling. A prominent feature of this remodelling is an increase in the number and size of the blood vessels- formed from pre-existing capillaries – angiogenesis (Siddiqui et al., 2007; Wilson, 2003). This is triggered by many different endogenous angiogenic stimulators such as vascular endothelial growth factor (VEGF), and inhibited by endogenous angiogenic inhibitors such as tumstatin. Tumstatin is the non-collagenous domain (NC1) of the collagen IV α3 chain which, when cleaved, inhibits endothelial cell proliferation and induces apoptosis. Experiments described in this thesis have for the first time demonstrated the absence of tumstatin in the airways of individuals with asthma and lymphangioleiomyomatosis (LAM) as well as the functional responses to tumstatin as an angiogenic inhibitor, both in vitro and in vivo, in the airway. Although tumstatin was absent from the airways of asthmatic and LAM individuals it was present in the airways of individuals with no airways disease, chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. No significant difference was seen in the levels of the Goodpasture Binding Protein (GPBP), a phosphorylating protein responsible for the alternate folding of tumstatin, between asthmatic, LAM and individuals with no airways disease. The αvβ3 integrin, reported to be necessary for the activity of tumstatin, as well as the individual αv and β3 sub-units were shown to be equally expressed in the airways of all patient groups. Co-localisation of tumstatin, VEGF and the αvβ3 integrin was seen in the disease free airways, however, a different pattern of VEGF and the αvβ3 integrin expression was observed in asthmatic and LAM airways with minimal co-localisation. Tumstatin was detected in serum and bronchoalveolar lavage fluid (BAL-f) samples from asthmatics and individuals with no airway disease, however there was no significant difference in the level of expression between the two groups. It was demonstrated that the tumstatin detected in the serum and BAL-f samples from asthmatics and individuals with no airway disease was part of the whole collagen IV α3 chain and not in its free and potentially active form. The ability of recombinant tumstatin to inhibit tube formation and proliferation of primary pulmonary endothelial cells was demonstrated for the first time. Further, the functional response of tumstatin was demonstrated in vivo in a mouse model of allergic airway disease. Tumstatin inhibited angiogenesis in the airway and decreased airway hyperresponsiveness. Whether there is potential for tumstatin, or a derivative thereof, to be of therapeutic value in airways diseases in which angiogenesis is a component should be the subject of future studies.

Thesis (M.D.S.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 106-152) Also available in print.

Objectives: To investigate whether any changes in nasal cavity dimensions or subjective report of nasal symptoms exist after rapid maxillary expansion using two types of expansion appliances, comparing results with an untreated control group. Methods: Subjects were randomly assigned into one of three groups: tooth-borne or bone-anchored expander or untreated control. Acoustic rhinometry was used to measure minimal cross-sectional area and volume of the nasal cavity over three timepionts for treatment subjects and two timepoints for control subjects, taken along with the NOSE Instrument survey. Results: No significant changes in nasal cavity dimension or subjective reports were found in subjects treated with tooth- or bone-anchored appliances compared to control subjects over three timepoints. In addition, non-significant correlation was observed between nasal airway dimensional change and subject symptoms. Conclusions: Rapid maxillary expansion does not result in change of i) nasal airway dimensions or ii) the sensation of nasal symptoms. / Orthodontics

Background: Fraction of exhaled nitric oxide (FENO) is a promising non-invasive index of airway inflammation that may be used to assess respiratory effects of air pollution. We evaluated FENO as a measure of airway inflammation after controlled exposure to diesel exhaust or ozone. Methods: Healthy volunteers were exposed to either diesel exhaust (particle concentration 300 mu g/m(3)) and filtered air for one hour, or ozone (300 ppb) and filtered air for 75 minutes. FENO was measured in duplicate at expiratory flow rates of 10, 50, 100 and 270 mL/s before, 6 and 24 hours after each exposure. Results: Exposure to diesel exhaust increased FENO at 6 hours compared with air at expiratory flow rates of 10 mL/s (p = 0.01) and at 50 mL/s (p = 0.011), but FENO did not differ significantly at higher flow rates. Increases in FENO following diesel exhaust were attenuated at 24 hours. Ozone did not affect FENO at any flow rate or time point. Conclusions: Exposure to diesel exhaust, but not ozone, increased FENO concentrations in healthy subjects. Differences in the induction of airway inflammation may explain divergent responses to diesel exhaust and ozone, with implications for the use of FENO as an index of exposure to air pollution.

Introduction: Asthma is a disease that occurs worldwide and approximately 300 million people carry this disease. It is characterized by chronic inflammation, airway obstruction and airway hyper-responsiveness (AHR). This T-lymphocyte controlled disease has symptoms such as coughing, wheezing, and chest tightness. In addition to chronic inflammation, asthma is also caused by overproduction of mucus and airway wall remodelling. The chronic inflammation and airway wall remodelling are suggested to contribute to the AHR and airway obstruction. AHR is a way to measure the reactivity in the airways in asthmatics. IL-13 has been shown to play an important role in the development of AHR, and biopsies from bronchial submucosa and air way smooth muscle (ASM) in humans have shown an increased concentration of IL-13 in severe asthma. Aim: The aim of this work was to evaluate if IL-13 is able to enhance the 5-HT response in mouse tracheal segments, which had been cultured for 2 days and, if so, try to unravel the underlying mechanism for this phenomenon. Literature reports that IL-13 enhanced contractions in mouse trachea in presence of KCl and CCH. Earlier work within this project did not find any clear proof for this observation. However, in this work this observation will be evaluated in a more controlled fashion by correcting for size and location of the trachea. Methods: The trachea was removed from Balp/c mice and cultured in small wells for two days in DMEM medium and various additions were performed to the medium for understanding the effect of e.g. IL-13 on the cells. The contractility change due to IL-13 and various additions in segments challenged with KCL, CCH and 5-HT were measured in a tissue-organ bath. Results and Conclusion: A more enhanced CCH induced contraction of IL-13 treated segments was obtained for the lower part compared to the upper part of the trachea. IL-13 enhanced the response in the ASM to 5-HT after two days of culturing. An increased concentration of the cytokine IL-13 in the airways from TH2-cells enhances the reactivity to 5-HT in the ASM. The underlying mechanism might involve JNK and ERK but more experiments are needed to statistically ensure this claim.

In mammals, the neurogenic inflammatory response can be induced by stimulation or activation on the peripheral sensory C-fibers to release neuropeptides from the peripheral terminals, at the same time their afferent functions are enhanced. There are several neuropeptides stored and released from peripheral terminals of the afferent fibers, such as substance P (SP), neurokinin A, and calcitonin gene related peptide (CGRP). SP is one of the major inflammatory mediators of neurogenic inflammation that can act on neurokinin-1 receptors on smooth muscles and endothelial cells of blood vessels, causing vasodilatation, endothelial gap formation, and local plasma leakage. There are many studies and reports indicate that animals pretreated with a short period non-lethal hyperthermia can induce heat shock response and activate the expression of a group of inducible proteins called heat shock proteins (HSPs), and this stress response reduces the injury by same or other following stresses. In this study, the hyperthermia treatment (HT) was implemented by 42¢J hot water bath and the core body temperature of anesthetized rat was elevated and maintained around 42.0¡Ó0.5¢J for 15 min, and the normothermia control treatment (NT) was implemented by 37¢J warm water bath with the same period. 24 hours after NT or HT, the neurogenic plasma leakage was induced by intravascular injection with capsaicin (90 £gg/kg), SP (3 £gg/kg), or electrical stimulation on the right thoracic vagus nerve. The blood pressures of each animal were continually recorded during the neurogenic inflammation induction or sham operation. The amount of neurogenic inflammation of airway was evaluated by the area density leaky blood vessels. The leaking vessels were labeled with India ink and quantitative analysis by morphometric method. Plasma leakage was also measured by interstitial Evans blue concentration. The results indicated that HT could reduce plasma leakage and hypotension of the neurogenic inflammation that induced by capsaicin, SP or electrical stimulation on vagus nerve. Animals pretreated with aminoguanidine (a selective inhibitor of iNOS) had no significant effect on the neurogenic inflammation by following systemic SP infusion, but that could eliminate the anti-neurogenic inflammatory effect of HT. Animal applied with diphenhydramine (an antagonist of histamine H1 receptor) could attenuate the neurogenic inflammation by following systemic SP infusion, and HT could attenuate the neurogenic inflammation that with or without H1 receptor antagonist. This result indicates that NO synthesis and the activity of iNOS have few effects on neurogenic inflammation of airway, but it plays a critical factor on the initiation of heat shock response. The neurogenic inflammation induced by SP not only direct act on blood vessels but have other indirect effect by the histamine H1 receptor to enhance inflammation. Neonatal rats received high dose capsaicin treatment would induce irreversible sensory C-fiber denervation. The adult rats that were neonatally treated with capsaicin showed a more serious inflammatory response to systemic SP infusion as compared with animals neonatally treated with vehicle. HT still had the anti-inflammatory effects on the neurogenic inflammation that induced by SP. The results indicated that animals with sensory C-fiber denervation might conserve their neurogenic inflammatory responses and were hypersensitive to SP. In conclusion, the HT could attenuate the neurogenic inflammation that induced by different drugs or methods, and the anti-inflammatory effects were correlated with the increase in HSP72 expression. In the neurogenic inflammation induced by SP, the activation of histamine H1 receptors may enhance inflammation, but the activity of endogenous iNOS was less effective.

Hematopoietic cells of the myelomonocytic lineage play a central role in orchestrating both innate and adaptive immunity. They are important in the control of infectious agents and in the pathogenesis of diseases characterized by dysregulated immune response. Like allergic asthma in human patients, recurrent airway obstruction (RAO) of horses is a disease exemplified by chronic airway inflammation in the absence of infectious agents. However, unlike allergic asthma, RAO is marked by preponderance of neutrophils rather than eosinophils in the airways. Attempts to understand the immunological basis of RAO by studying lymphocytes produced equivocal results. This thesis examined the possible role of alveolar macrophages (AM) recovered from bronchoalveolar lavage fluid (BALF) in RAO. Since macrophages are predominantly derived from circulating monocytes, the thesis investigated first the phenotypic characteristics of circulating monocytes, second those of macrophages in vitro derived from monocytes, and finally attributes of AM derived in vivo. Flow cytometric analysis following antibody staining of monocytes from 61 horses showed that the clustering pattern of human leukocytes may not always be extrapolated to horses when using this technique since clusters of granulocytes often spill over into the monocyte population. The study showed that DH24A, a monoclonal antibody directed against CD90, which recognizes T cells in other species, will specifically recognize granulocytes in horses and was therefore used to separate neutrophils from monocytes during analysis. In addition, investigation of circulating monocytes showed that expression of the hemoglobin-haptoglobin receptor CD163 on circulating monocytes is significantly increased in horses with systemic inflammation when compared with healthy horses. Evaluating cytokine and chemokine production by macrophages, it was demonstrated that CD163+ macrophages preferentially expressed IL10 while CD163- macrophages showed predominant expression of CCL17. It was, therefore, concluded that CD163+ IL10-producing macrophages of horses are homologues of the alternatively activated anti-inflammatory macrophage subset of humans. Finally, probing of alveolar macrophages for CD163 and CD206 expression showed a significant reduction in the proportion of CD163+ macrophages in horses with RAO. These findings suggest that RAO is associated with a reduction in anti-inflammatory macrophages, an observation that may in part explain the chronic airway inflammation associated with this disease.

No description available.

Aberrant phenotypes of airway smooth muscle cells are central to the pathophysiology of asthma. The hypercontractile nature of these cells and hypertrophy are the key reasons for the excessive narrowing of the airways observed in allergic asthma. Although previous studies have indicated a role of enhanced content of smMLCK in modulating the contractile reactivity, as well as an indication of hypertrophy of HASM cells in asthmatic conditions, the effect of IgE on the expression of smMLCK in HASM cells is not fully understood. In this study, we demonstrate that IgE augments the expression of smMLCK at the mRNA and protein level. Inhibition of IgE binding with anti-FcεRI blocking antibody, Syk silencing, pharmacological inhibitors to MAPK (ERK1/2, p38, and JNK) and PI3K significantly diminished the IgE-mediated smMLCK expression in HASM cells. Finally, we found that IgE, similar to metacholine induces the contraction of HASM cells grown on collagen gel matrix. Our data suggest that IgE stimulates the phosphorylation of ERK, P38, STAT3 and induces the dephosphorylation of smMLCK to phosphorylate myosin regulatory light chain in HASM cells. Taken together, our data suggest a modulatory role of IgE in regulating the contractile machinery and hypertrophic phenotype of HASM cells.