<p>Cysteinyl leukotrienes (LTC4 , D4 , E4 ) play an important role in the pathophysiology of asthma. They produce bronchoconstriction, microvascular leakage and eosinophil infiltration into the airway mucosa. They mediate the airway responses following allergen inhalation and exercise. This thesis examined new effects of cysteinyl leukotrienes on three cells in human airways: an antigen presenting cell (dendritic cell), a key inflammatory cell (eosinophil) and a structural cell (smooth muscle). Dendritic cells initiate allergen-induced airway responses by presenting the allergen to lymphocytes. Cysteinyl leukotrienes are necessary for the migration of dendritic cells from tissues to the regional lymph nodes. We observed that they also regulate the recruitment of myeloid dendritic cells from peripheral blood following an allergen inhalation. In a clinical trial, we observed that two weeks of treatment with a leukotriene receptor antagonist prevented the allergen-induced decrease in the number of circulating myeloid, but not plasmacytoid, dendritic cells in atopic asthmatic subjects. This was in keeping with our observation that greater proportion of myeloid dendritic cells than plasmacytoid dendritic cells expressed the CysLT1 receptor. Esoinophilic infiltration of the airway is a characteristic feature of asthma pathology. We observed that inhalation of cysteinyl leukotrienes caused an increase in the number of eosinophils in airway mucosa and lumen in subjects with atopic asthma. We also observed that leukotriene E4 caused greater eosinophilia than leukotriene D4 . We further examined bone marrow eosinophilopoiesis as one of the mechanisms by which leukotrienes cause airway eosinophilia. Treatment of atopic asthmatic subjects with a leukotriene-receptor antagonist for two weeks attenuated allergen-induced increase in the number of eosinophl/basophil colony forming units in the bone marrow. This was the first in-vivo observation of a direct role of cysteinyl leukotrienes in regulating eosinophilopoiesis in humans. The first recognized biological effect of cysteinyl leukotrienes was their ability to contract smooth muscles. We observed that they could also modulate another property of human airway smooth muscle, i.e., migration. We observed that human airway smooth muscle cells show chemotaxis towards platelet-derived growth factor. Although leukotrienes by themselves were not chemoattractants, they caused chemokinesis of smooth muscle cells and augmented chemotaxis towards platelet-derived growth factor. A leukotriene receptor antagonist inhibited this. The mechanism was not dependent on increased integrin expression on smooth muscle, or Src-kinase or phosphatidylinositol 3-kinase phosphorylation. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p>Airway hyperresponsiveness (AHR) is a characteristic feature of asthma. However, the mechanisms underlying AHR are complex and likely to be multi-factorial. A number of animal models have focused on understanding the mechanisms of transient airway inflammation and the associated transient airway dysfunction that occur following brief allergen exposure. While much has been learned about the role of acute inflammation in this brief allergen-induced response, a limitation of these studies is that the airway dysfunction is not fully representative of the AHR present in asthma. For example, we have shown that effective anti-inflammatory treatment results in only a modest reduction in AHR, indicating that AHR is usually sustained in asthma. This suggests that other mechanisms, including airway remodeling, likely play an important role in the pathophysiology of AHR. The subsequent focus of this thesis has been firstly, to demonstrate that sustained airway dysfunction develops in mice chronically exposed to allergen, and secondly, to explore the potential mechanisms and associations underlying this phenomenon. By subjecting sensitized mice to either brief or chronic periods of allergen exposure, we have developed a novel model in which chronic allergen exposure results in sustained airway dysfunction and structural changes of the airway. Subsequently, mice deficient for IL-4, IL-5 or IL-13 were studied using similar protocols. IL-4 and IL-13, but not IL-5, are critical for the development of airway remodeling, and in the absence of remodeling, mice were protected from developing sustained airway dysfunction. In further experiments, mice were either T cell immuno-depleted using monoclonal antibodies, or were treated with an anti-IL-13 fusion protein. When these interventions were given after chronic allergen exposure, at a time when airway remodeling was established, neither intervention attenuated the sustained airway dysfunction. Taken together, these results provide new information about basic physiological mechanisms underlying the sustained airway dysfunction present in asthma.</p> / Doctor of Philosophy (PhD)

<p>Asthma is a chronic airways disease characterised by recurrent episodes of wheezing and variable airflow obstruction, which is usually reversible spontaneously or with treatment, and with airway hyperresponsiveness, and airway inflammation. Allergen inhalation by sensitized, atopic asthmatics enhances airway hyperresponsiveness and inflammation, providing a useful model to study allergic asthma. The aim of this thesis was to characterise the allergen-induced changes in bone marrow eosinophil/basophil (Eo/B) progenitors, their ability to migrate out of the bone marrow and to determine whether these cells accumulate in the airways to participate in the allergen-induced airway inflammatory response. In addition, this thesis attempted to investigate the importance of pro- and anti-inflammatory cytokines and chemokines which contribute to the migration and differentiation of these cells. Bone marrow aspirates, blood and sputum samples were obtained from subjects at various time points both before and following allergen inhalation challenge. In comparison to isolated early responders, who develop no allergen-induced late asthmatic responses or airway hyperresponsiveness, bone marrow Eo/B progenitor numbers were increased in dual responders, who develop marked allergen-induced airway eosinophilia and airway hyperresponsiveness. Increases in IL-3-responsive progenitors were detected as early as 5 hours post-allergen, and IL-5-responsive progenitors at 12 and 24 hours post-allergen in dual responders only. Bone marrow, blood and sputum IL-5 protein levels increased at 12 and 24 hours in dual responders only and these increases correlated with increases in IL-5-responsive progenitors. Additionally, bone marrow IFN-γ levels increased in dual responders at 48 hours coinciding with decreases in bone marrow EoB progenitors. Expression of the receptor for eotaxin (CCR3) was detected on primitive (CD34 immunopositive cells) and eosinophil-lineage committed progenitors (CD34 + IL-5Rα+ cells) by flow cytometry and confirmed by co-localization of CCR3 messenger RNA to CD34+ cells using in-situ hybridization. When pre-allergen was compared to 24 hours post-allergen levels, significant increases in bone marrow CD34+ CCR3 + cells were detected in dual responders, who also developed a significant sputum and blood eosinophilia and increased methacholine airway responsiveness. In contrast, a significant attenuation of bone marrow CD34+ CCR3 + cells was observed in isolated early responders. In a dose dependent fashion eotaxin, but not IL-5, stimulated CD34+ progenitor cell migration in vitro . (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

Cigarette smoke (CS) has many damaging effects on the body, and the chronic inhalation of cigarette smoke can change immunological functions through impact on both innate and adaptive immunity. The incidences of many diseases are affected by the adverse effects of cigarette smoke on the immune system, and the induction of an inflammatory response, which affects several tissues and organs. On this basis, a comparison of smokers′ and non-smokers′ immunoglobulin levels could provide valuable insights into the mechanisms of smoking related diseases. Although the effects of cigarette smoking on humoral and cellular immunity have been investigated previously, the results have varied between the studies, and therefore more research is still required. The aim of this study was to determine whether the levels of immunoglobulin (Ig) isotypes are different in the serum and saliva of non-smoking individuals compared to smoking individuals. An examination of serum and saliva would provide information on the effects of cigarette smoke systemically and in the oral mucosa, respectively. The effects of cigarette smoke extract on B-cell secretions were also examined to establish whether cigarette smoke components can have direct effects on immunoglobulin production by B cells. In order to determine Ig isotype levels, antibody microarray techniques were established and calibrated for determining the sample concentrations of IgM, IgG, IgA and IgD. The results showed that smoking has different effects on systemic and salivary immunoglobulin levels. In the serum, smokers had decreased levels of IgG and IgD, but increased IgM and IgA levels compared to non-smokers. However, in the saliva smokers had decreased levels of IgG, IgD, and IgM, whereas there were increased levels of IgA in smokers’ saliva. As CS has been found to influence the serum and salivary levels of Ig isotypes ex-vivo, the mechanisms underlying these effects were investigated in vitro to determine whether the changes were as a result of a direct effect of the CS on B-cells. This study has shown that CS had deleterious effects on the production and the levels, of Ig isotypes. These results support the concept that CS is related to diseases, and more research is necessary in this field.

Assessment of the microcirculation is of major importance in understanding the physiology of the vasculature and in assessing te vascular effects of pathological conditions such as diabetes, hypertension and sepsis. Transdermal iontophoresis can be used to non‐invasively introduce vasoactive drugs into the skin. The response to these drugs of the local cutaneous microvasculature can be measured by laser Doppler flowmetry methods. Although these techniques have been used together for over two decades, there are still important methodological issues to be resolved. This work is aimed at optimizing transdermal iontophoresis as a tool for microvascular assessment by focusing on the main methdological issues: non‐specific vasodilatation, drug delivery protocols and analysis of blood flow data. Non‐specific vasodilatation, an increase blood flow during iontophoresis of non‐vasoactive compounds, is an important problem as it interferes with the response to the administered drug. By investigating this effect in healthy volunteers, we found that the extent of the non‐specific response differs between the positive and negative electrode and that it is dependent on the voltage over the skin andon the ionic strength of the vehicle in which the drug is dissolved. We also found that the extent of the non‐specific response could be reduced by applying local anesthetics and by pre‐treatment with antihistamine drugs. These results suggest that non‐specific effects could be mediated by depolarization or hyperpolarisation of cells, triggering neural and histamine related mechanisms that finally lead to vasodilatation of the local microvasculature. To prevent non‐specific effects from occurring during the experiments, our results show that the current strength and the total electric charge during iontophoresis should be limited to 0.02 mA and12 mC, respectively. Furthermore, drug solutions at physiological ionic strengths should be used. Under these conditions, adequate responses to the most commonly used drugs, acetylcholine (ACh) and sodium nitroprusside (SNP), are obtained while no significant non‐specific vasodilatation occurs. The results of our investigations show that blood responses to ACh and SNP applied by a single iontophoretic pulse can well be escribed by conventional dose‐response models, which enables a more powerful analysis and comparison between drugs or possibly patient groups as compared with conventional aalysis methods. Finally, we have incorporated drug transport and physiological response to the local drug concentration during iontophoresis of vasoactve drugs into a single model. Validation of this model using measured responses to ACh and SNP shows that the commonly used assumption that the local drug concentration during iontophoresis is linearly proportional to the electric charge may not be valid. / Mikrocirkulationen, som inbegriper kroppens minsta blodkärl, transporterar syre och näringsämnen till våra celler. Vissa sjukdomar, som diabetes, hjärt‐kärlsjukdom och akut blodförgiftning leder till förändringar hos mikrocirkulationen. Mekanismerna bakom dessa förändringar är delvis okända. Det finns därför ett stort behov av kliniska mättekniker som kan bedöma mikrocirkulationens funktion. Vid jontofores placeras en elektrod tillsammans med ett läkemedel på huden. När en svag elektrisk ström anbringas transporteras läkemedlet ner genom hudlagren. Effekterna av ett kärlaktivt läkemedel som appliceras på detta sätt kan sedan avläsas non‐invasivt med laser Doppler‐teknik. En stor fördel med jontoforesmetoden, förutom att den är non‐invasiv, är att läkemedelsdoserna som tillförs kroppen är mycket små och därmed ger de inte upphov till några systemiska bi‐effekter. I avhandlingen presenteras forskning, vilkas målsättning är att lösa några av de viktiga frågorna kring transdermal jontofores så att tekniken optimeras för att denskall kunna brukas som ett verktyg vid kliniska undersökningar av mikrocirkulationen. Den första delen ägnas ett fenomen som kallas ospecifik vasodilatation. Det uppstår vid jontofores av substanser som är inte kärlaktiv, som vatten och koksaltlösning. Resultaten från dessa försök indikerar att den ospecifika vasodilatationen beror på framför allt spänningen över huden, vilken i sin tur är relaterad till jon‐koncentrationen hos läkemedelslösningen. Vidare registreras att mekanismen bakom den ospecifika vasodilatationen delvis är neuralt medierad genom att de till stor del år att förhindra med hjälp av lokal bedövning. Dessutom leder förbehandling med anti‐histamina läkemedel till minskade ospecifika reaktioner, vilket också indikerar att lokala inflammatoriska processer är inblandande. Den andra delen av avhandlingen ägnas att optimera försöksprotokollen för jontofores. Till att börja med utvecklas ett protokoll som ger ett adekvat läkemedelssvar samtidigt som ospecifika effekter minimeras. Det visar sig är möjligt genom att begränsa strömstyrkan och den elektriska laddningen under jontoforesen och genom att använd läkemedelslösningar som har en fysiologisk jonstyrka. Resultaten visar också att blodflödesförändringen som registreras under jontofores av acetylkolin och natriumnitroprussid kan eskrivas med hjälp av konventionella dos‐responsmodeller, vilket möjliggör en mer exakt analys av det mikrocirkulatoriska svaret samt underlättar jämförelse mellan olika läkemedel elle patientgrupper. Slutligen presenteras en mekanistisk model för det mikrocirkulatoriska svaret vid jontofores. Modellen beskriver läkemedlets transport från elektroden ner genom huden, clearance i huden vilken beror på diffusion och det lokala blodflödet, samt förändringen i blodflöde som sker på grund av läkemedlet. Modellen valideras genom försök på försökspersoner och resultaten visar att förändringarna i blodflödet åstadkommet av acetylklin och natriumnitroprussid med denna modell kan beskrivas på ett exakt sätt. Vidare visar resultaten att det sker en betydande clearance av läkemedel i huden under jontofores. Detta har väsentlig betydelse när man ska uppskatta den lokala jontoforesdosen. / The author changed surname from Droog to Tesselaar in January 2006.

<p>The alpha-2 adrenergic receptor (α₂-AR) subfamily includes three different subtypes, α_2A-, α_2B- and α_2C-AR, all believed to exert their function through heterotrimeric G_i/o-proteins. The present study was undertaken in order to investigate subtype differences in terms of cellular response and to explore other potential signalling pathways of α₂-ARs.</p><p>Evidence is provided for a strong G_s-protein coupling capability of the α_2B-AR, leading to stimulation of adenylyl cyclase (AC). The difference between the α_2A- and α_2B-AR subtypes, in this respect, was shown to be due to differences in the second intracellular loops of the receptor proteins. Substitution of the second loop in α_2A-AR with the corresponding domain of α_2B-AR enrolled the chimeric α_2A/α_2B receptor with functional α_2B-AR properties. Dual G_i and G_s coupling can have different consequences for AC output. Using coexpression of receptors and G-proteins, it was shown that the ultimate cellular response of α_2B-AR activation is largely dependent on the ratio of G_i- to G_s-protein amounts in the cell. Also G_i- and G_o-proteins appear to have different regulatory influences on AC. Heterologous expression of AC2 together with G_i or G_o and the α_2A-AR resulted in receptor-mediated inhibition of protein kinase C-stimulated AC2 activity through G_o, whereas activation of G_i potentiated the activity. </p><p>α₂-ARs mobilize Ca²⁺ in response to agonists in some cell types. This response was shown to depend on tonic purinergic receptor activity in transfected CHO cells. Elimination of the tonic receptor activity almost completely inhibited the Ca²⁺ response of α₂-ARs.</p><p>In conclusion, α₂-ARs can couple to multiple G-proteins, including G_i, G_o and G_s. The cellular response to α₂-AR activation depends on which receptor subtype is expressed, which cellular signalling constituents are engaged (G-proteins and effectors), and the signalling status of the effectors (dormant or primed).</p>

The alpha-2 adrenergic receptor (α2-AR) subfamily includes three different subtypes, α2A-, α2B- and α2C-AR, all believed to exert their function through heterotrimeric Gi/o-proteins. The present study was undertaken in order to investigate subtype differences in terms of cellular response and to explore other potential signalling pathways of α2-ARs. Evidence is provided for a strong Gs-protein coupling capability of the α2B-AR, leading to stimulation of adenylyl cyclase (AC). The difference between the α2A- and α2B-AR subtypes, in this respect, was shown to be due to differences in the second intracellular loops of the receptor proteins. Substitution of the second loop in α2A-AR with the corresponding domain of α2B-AR enrolled the chimeric α2A/α2B receptor with functional α2B-AR properties. Dual Gi and Gs coupling can have different consequences for AC output. Using coexpression of receptors and G-proteins, it was shown that the ultimate cellular response of α2B-AR activation is largely dependent on the ratio of Gi- to Gs-protein amounts in the cell. Also Gi- and Go-proteins appear to have different regulatory influences on AC. Heterologous expression of AC2 together with Gi or Go and the α2A-AR resulted in receptor-mediated inhibition of protein kinase C-stimulated AC2 activity through Go, whereas activation of Gi potentiated the activity. α2-ARs mobilize Ca2+ in response to agonists in some cell types. This response was shown to depend on tonic purinergic receptor activity in transfected CHO cells. Elimination of the tonic receptor activity almost completely inhibited the Ca2+ response of α2-ARs. In conclusion, α2-ARs can couple to multiple G-proteins, including Gi, Go and Gs. The cellular response to α2-AR activation depends on which receptor subtype is expressed, which cellular signalling constituents are engaged (G-proteins and effectors), and the signalling status of the effectors (dormant or primed).

<p>The gastrointestinal mucus gel layer and blood flow are two important mechanisms for protection at the pre-epithelial and sub-epithelial levels, respectively. <i>Helicobacter pylori</i> might circumvent these mechanisms and elicit a chronic inflammatory response with consequent ulcers in the stomach and duodenum. In this thesis, the physical state and properties of the adherent mucus gel layer was studied from the stomach to colon. Furthermore, the acute and chronic effects of <i>H. pylori</i> on the integrity of the mucus gel layer and mucosal blood flow were studied in the anesthetized rat.</p><p>A translucent mucus gel covers all studied segments of the gastrointestinal tract during fasting conditions, with the thickest layers in the colon and ileum. Carefully applied suction revealed that the mucus gel was a multi-layered structure comprising a firmly adherent layer covering the mucosa, impossible to remove, and a loosely adherent upper layer. The firmly adherent layer was thick and continuous in the corpus (80μm), antrum (154μm) and colon (116μm), but thin (<20μm) and discontinuous in the small intestine.</p><p>Following mucus removal, a rapid renewal of the loosely adherent layer ensued. The highest rate was observed in the colon with intermediate values in the small intestine. Mucus renewal in the stomach was attenuated on acute luminal application of water extracts from <i>H. pylori</i> (HPE). In animals with a chronic <i>H. pylori</i> infection the mucus renewal rate was unaffected, but the total gastric mucus gel thickness was reduced and the mucus secretory response to luminal acid (pH1) attenuated in the antrum. </p><p>HPE from type I strains acutely reduced corporal mucosal blood flow, measured with laser-Doppler flowmetry, by approximately 15%. The reduction in blood flow was mediated by a heat stable factor other than VacA and CagA. Inhibition of endogenous nitric oxide production with Nω-nitro-l-arginine augmented the decrease. However, ketotifen, a mast cell stabilizer, completely attenuated the effect of the extract as did the platelet activating factor (PAF) receptor-antagonist, WEB2086, thus depicting a detrimental role for the microvascular actions of PAF.</p>

The gastrointestinal mucus gel layer and blood flow are two important mechanisms for protection at the pre-epithelial and sub-epithelial levels, respectively. Helicobacter pylori might circumvent these mechanisms and elicit a chronic inflammatory response with consequent ulcers in the stomach and duodenum. In this thesis, the physical state and properties of the adherent mucus gel layer was studied from the stomach to colon. Furthermore, the acute and chronic effects of H. pylori on the integrity of the mucus gel layer and mucosal blood flow were studied in the anesthetized rat. A translucent mucus gel covers all studied segments of the gastrointestinal tract during fasting conditions, with the thickest layers in the colon and ileum. Carefully applied suction revealed that the mucus gel was a multi-layered structure comprising a firmly adherent layer covering the mucosa, impossible to remove, and a loosely adherent upper layer. The firmly adherent layer was thick and continuous in the corpus (80μm), antrum (154μm) and colon (116μm), but thin (&lt;20μm) and discontinuous in the small intestine. Following mucus removal, a rapid renewal of the loosely adherent layer ensued. The highest rate was observed in the colon with intermediate values in the small intestine. Mucus renewal in the stomach was attenuated on acute luminal application of water extracts from H. pylori (HPE). In animals with a chronic H. pylori infection the mucus renewal rate was unaffected, but the total gastric mucus gel thickness was reduced and the mucus secretory response to luminal acid (pH1) attenuated in the antrum. HPE from type I strains acutely reduced corporal mucosal blood flow, measured with laser-Doppler flowmetry, by approximately 15%. The reduction in blood flow was mediated by a heat stable factor other than VacA and CagA. Inhibition of endogenous nitric oxide production with Nω-nitro-l-arginine augmented the decrease. However, ketotifen, a mast cell stabilizer, completely attenuated the effect of the extract as did the platelet activating factor (PAF) receptor-antagonist, WEB2086, thus depicting a detrimental role for the microvascular actions of PAF.