過敏性疾病（如過敏性哮喘和過敏性皮炎）發病率在香港及世界均呈上升趨勢。過敏性哮喘是一種慢性反復發作的炎症疾病，而過敏性皮炎是一種慢性皮膚炎症。呼吸道細菌及金黃色葡萄球菌可分別加重過敏性哮喘病人氣道炎症及過敏性皮炎患者的炎症反應。人體對細菌的先天免疫反應主要通過模式識別受體(PRR)介導。NOD樣受體(NLR)和Toll樣受體(TLR)是兩種重要的PRR。 NLR 家族成員NOD2幾乎識別所有細菌中結構保守的胞壁醯二肽(MDP)。而LTR2識別範圍廣泛的病原相關分子模式，如革蘭氏陽性菌中的肽聚糖(PGN)和脂磷壁酸(LTA)，以及人工合成的脂蛋白Pam3CSK4。 / 本研究包括：NOD2配體MDP，哮喘相關的腫瘤壞死因子家族成員LIGHT對共培養的人嗜鹼性粒細胞和人支氣管上皮細胞的活化作用；熱滅活的金黃色葡萄球菌（HKSA），MDP，TLR2配體PGN，LTA，以及Pam3CSK4對共培養的人嗜鹼性粒細胞和人皮膚成纖維細胞的活化作用；在體內NLR配體對卵清蛋白(OVA)致敏的哮喘小鼠的作用。 / 研究發現，在共培養體系中，MDP能顯著增強嗜鹼性粒細胞與支氣管上皮細胞表面粘附因子（細胞間粘附因子ICAM-1 及血管細胞粘附因子VCAM-1）的表達。同時，MDP能顯著促進共培養體系中炎症相關細胞因子IL-6，趨化因子CXCL8及抗菌肽β-防禦素2的釋放。在MDP刺激下，支氣管上皮細胞是共培養體系中釋放IL-6，CXCL8及β-防禦素2的主要細胞。在MDP刺激下，嗜鹼性粒細胞中包括胞核因子-kappaB（NF-κB）在內的幾個核轉錄因子的表達上升。ICAM-1，VCAM-1，IL-6，CXCL8，及β-防禦素2的表達被信號分子化學抑制劑所抑制，結果表明，嗜鹼性粒細胞與支氣管上皮細胞的相互作用受不同的信號通路（NF-κB, p38 MAPK 及 JNK）調節。OVA致敏小鼠實驗表明，NLR配體能增加分泌粘蛋白的杯狀細胞在肺氣管中的數量，使小鼠支氣管下皮結締組織纖維化並增厚。NLR配體進而提高過敏性哮喘小鼠支氣管肺泡灌洗液中CCL5與IL-13 的表達水平。 / 研究表明，在嗜鹼性粒細胞和皮膚成纖維細胞的共培養體系中，HKSA，MDP，PGN，LTA，或Pam3CSK4顯著誘導ICAM-1, IL-6, CXCL8, CCL2 和 CCL5 的表達。而嗜鹼性粒細胞與皮膚成纖維細胞的直接相互作用是釋放IL-6, CXCL8, CCL2 與 CCL5 所必需的。嗜鹼性粒細胞與皮膚成纖維細胞的相互作用並釋放細胞因子與趨化因子受p38 MAPK 及 NF-κB信號通路調控。 / 在嗜鹼性粒細胞與支氣管上皮細胞共培養體系中，LIGHT 可能通過受體HVEM 與 LTβR顯著增強支氣管上皮細胞表面粘附因子的表達，提高細胞因子IL-6, CXCL8 與 MMP-9的釋放。 / 研究結果表明，在過敏炎症中，通過與組織細胞（如支氣管上皮細胞，人皮膚成纖維細胞）相互作用，嗜鹼性粒細胞有利於組織細胞對病原相關的分子模式作出反應。因此，研究結果對細菌介導的先天性免疫應答與過敏炎症的加重之間的聯繫作出了新的解釋。以上結果也增強了我們對LIGHT在氣道重塑中的免疫病理作用及其作為氣道重塑治療靶標的認識。 / The incidences of allergic diseases such as allergic asthma and atopic dermatitis (AD) are increasing in Hong Kong and worldwide. Allergic asthma is a chronically relapsing inflammatory pulmonary disease, while AD is a chronic inflammatory skin disorder. Respiratory bacterial and Staphylococcus aureus (S. aureus) infection can provoke allergen sensitization and subsequently amplify and sustain inflammation in allergic asthma and AD, respectively. The innate immune system recognizes bacterial infection through pattern recognition receptors (PRRs), two important PRRs involving in inflammatory and immune responses are nucleotide-binding oligomerization domain-like receptors (NLRs) and Toll-like receptors (TLRs). NOD2 is one member of the NLR family, which senses the conserved structural component muramyl dipeptide (MDP) in almost all bacteria. TLR2 recognizes a wide range of pathogen-associated molecular patterns (PAMPs) including peptidoglycan (PGN) and lipoteichoic acid (LTA) from Gram-positive bacteria and synthetic triacylated lipoprotein N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S] -lysine (Pam3CSK4). / In the present study, we investigated the effect of NOD2 ligand MDP, asthma-related tumor necrosis factor (TNF) family member LIGHT on human basophils co-cultured with human bronchial epithelial cells and the effect of heat-killed S. aureus, MDP, TLR2 ligands PGN, LTA and Pam3CSK4 on basophils co-cultured with human dermal fibroblasts, and the underlying intracellular mechanisms. The in vivo effect of NOD ligands on ovalbumin (OVA)-sensitized allergic asthmatic mice was also studied. / It was found that MDP could significantly enhance the cell surface expression of adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on basophils and primary human bronchial epithelial cells (HBE) in the co-culture system (all p < 0.05). MDP could further enhance the release of inflammatory cytokine interleukin (IL)-6, chemokine CXCL8, and epithelium derived anti-microbial peptide β-defensin 2 in the co-culture. HBE cells were the major source while basophils were the minor source to release IL-6, CXCL8 and β-defensin 2 in the co-culture upon MDP stimulation. The activities of several nuclear transcription factors, including NF-κB, were up-regulated in human basophils upon MDP stimulation. The cell surface expression of ICAM-1 and VCAM-1 and the release of IL-6, CXCL8 and β-defensin 2 were suppressed by the signaling molecule inhibitors, implying that the interaction between basophils and primary human bronchial epithelial cells could be differentially regulated by the NF-κB, p38 MAPK and JNK pathways. The animal study showed that iE-DAP and MDP could increase the number of mucin-secreting goblet cells, the thickness and fibrosis of the bronchial subepithelial tissue of airways from the OVA-sensitized mice. The iE-DAP and MDP could further promote the levels of CCL5 and IL-13 (all p < 0.05) in bronchoalveolar lavage fluid (BALF) of allergic asthmatic mice. / It was found that the induction of ICAM-1, IL-6, CXCL8, CCL2 and CCL5 was significantly promoted upon the interaction between human basophils and dermal fibroblasts activated by heat-killed S. aureus, MDP, PGN, LTA or Pam3CSK4. The release of IL-6, CXCL8, CCL2 and CCL5 might depend on the direct interaction of basophils and dermal fibroblasts. The p38 MAPK and NF-κB pathways should be involved in the release of the cytokines and chemokines upon the interaction of basophils and human dermal fibroblasts. / LIGHT could significantly promote the cell surface expression of adhesion molecule, the release of IL-6, CXCL8 and MMP-9 from human bronchial epithelial cells upon the interaction with basophils, probably through the receptors HVEM and LTβR. / The results suggest that, through the interaction with tissue-resident cells such as bronchial epithelial cells and dermal fibroblasts, basophils may facilitate the activation of tissue-resident cells in response to the PAMPs in allergic inflammation. The results therefore provide a new insight of the crucial link between the bacterial-mediated innate immune response and the exacerbation of allergic inflammation. The above results also enhance our understanding on the immunopathological roles of LIGHT in airway remodeling, and the potential therapeutic target for airway remodeling. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qiu, Huaina. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 165-196). / Abstract also in Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 摘要 --- p.ix / Publications --- p.xi / Table of Contents --- p.xiii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Asthma and atopic dermatitis (AD) --- p.1 / Chapter 1.2 --- Human basophils in allergic inflammation --- p.3 / Chapter 1.2.1 --- Development and morphology of basophils --- p.3 / Chapter 1.2.2 --- Receptors and products of basophils --- p.4 / Chapter 1.2.3 --- Cell surface markers on basophils --- p.7 / Chapter 1.2.4 --- Basophils in allergic inflammation --- p.7 / Chapter 1.3 --- Human bronchial epithelial cells in airway inflammation --- p.10 / Chapter 1.4 --- Human fibroblasts in AD --- p.11 / Chapter 1.5 --- Staphylococcus aureus (S. aureus) in AD --- p.12 / Chapter 1.6 --- NOD2 and TLR2 in allergic inflammation --- p.14 / Chapter 1.7 --- IL-33 in allergic inflammation --- p.18 / Chapter 1.8 --- IL-6 in allergic inflammation --- p.18 / Chapter 1.9 --- CXCL8 in allergic inflammation --- p.20 / Chapter 1.10 --- CCL2 in allergic inflammation --- p.21 / Chapter 1.11 --- CCL5 in allergic inflammation --- p.22 / Chapter 1.12 --- β-defensin 2 (HBD-2) in allergic inflammation --- p.23 / Chapter 1.13 --- ICAM-1 and VCAM-1 in allergic inflammation --- p.25 / Chapter 1.14 --- LIGHT and airway remodeling in allergic asthma --- p.25 / Chapter 1.15 --- Signal transduction pathways in allergic inflammation and pharmacological inhibitors --- p.26 / Chapter 1.15.1 --- Signal transduction pathways in allergic inflammation --- p.26 / Chapter 1.15.2 --- Signaling molecule inhibitors as new drugs for inflammatory diseases --- p.31 / Chapter 1.16 --- Aims and scope of the study --- p.32 / Chapter Chapter 2: --- Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Reagents and buffers for the purification of human basophils --- p.35 / Chapter 2.1.2 --- Primary cells and cell lines --- p.36 / Chapter 2.1.3 --- Heat-killed Staphyloccocus aureus (HKSA) --- p.38 / Chapter 2.1.4 --- Ligands for NLR and TLR2 --- p.39 / Chapter 2.1.5 --- Recombinant human cytokines --- p.39 / Chapter 2.1.6 --- Reagents and buffer solutions for flow cytometry --- p.40 / Chapter 2.1.7 --- RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR), and real-time quantitative PCR (qPCR) --- p.45 / Chapter 2.1.8 --- Cytometric Bead Array (CBA) Kits --- p.48 / Chapter 2.1.9 --- MILLIPLEX® MAP Human Cytokine/Chemokine Magnetic Bead Panel Kit --- p.49 / Chapter 2.1.10 --- Enzyme-linked immunosorbent assay (ELISA) kits --- p.49 / Chapter 2.1.11 --- Procarta Transcription Factor Assay kit --- p.50 / Chapter 2.1.12 --- Signal transduction inhibitors --- p.50 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Purification of primary human basophils and basophil culture --- p.50 / Chapter 2.2.2 --- Culture of KU812 cells --- p.51 / Chapter 2.2.3 --- Culture of primary human bronchial epithelial cells --- p.51 / Chapter 2.2.4 --- Culture of BEAS-2B cells --- p.52 / Chapter 2.2.5 --- Culture of human dermal fibroblasts --- p.52 / Chapter 2.2.6 --- Co-culture of primary human bronchial epithelial cells/human bronchial epithelial cell line (BEAS-2B) cells and basophils/KU812 cells --- p.52 / Chapter 2.2.7 --- Co-culture of human dermal fibroblasts and basophils/KU812 cells --- p.52 / Chapter 2.2.8 --- Co-culture of fixed primary human bronchial epithelial cells and basophils --- p.53 / Chapter 2.2.9 --- Co-culture of human dermal fibroblasts and basophils in the presence of transwell inserts --- p.53 / Chapter 2.2.10 --- CBA assay --- p.53 / Chapter 2.2.11 --- ELISA --- p.54 / Chapter 2.2.12 --- Human Transcription Factor Plex Assay --- p.54 / Chapter 2.2.13 --- Milliplex Human Cytokine / Chemokine Magnetic Panel assay --- p.54 / Chapter 2.2.14 --- Bio-Plex mouse cytokine assay --- p.55 / Chapter 2.2.15 --- Flow cytometric analysis of cell surface expression of target molecules --- p.55 / Chapter 2.2.16 --- Flow cytometric analysis of intracellular expression of target molecules --- p.55 / Chapter 2.2.17 --- Allergic asthmatic mice model --- p.57 / Chapter 2.2.18 --- Statistical analysis --- p.57 / Chapter Chapter 3: --- Muramyl Dipeptide Mediated Activation of Human Bronchial Epithelial Cells Interacting with Basophils: A Novel Mechanism of Airway Inflammation --- p.59 / Chapter 3.1 --- Introduction --- p.59 / Chapter 3.2 --- Results --- p.61 / Chapter 3.2.1 --- Cell surface expression of CD203c on basophils --- p.61 / Chapter 3.2.2 --- Intracellular expression of NOD2 protein --- p.63 / Chapter 3.2.3 --- Cell surface expression of adhesion molecules on basophils and primary human bronchial epithelial cells activated by MDP --- p.67 / Chapter 3.2.4 --- Induction of cytokine, chemokine and β-defensin 2 upon the interaction of basophils and bronchial epithelial cells stimulated by MDP --- p.71 / Chapter 3.2.5 --- Bronchial epithelial cells were the main source for the release of IL-6, CXCL8 and β-defensin 2 in co-culture --- p.74 / Chapter 3.2.6 --- Effects of signaling inhibitors on MDP-induced cytokines and adhesion molecules --- p.77 / Chapter 3.2.7 --- Differential activation of intracellular signaling pathways involved in the interaction of KU812 and BEAS-2B upon MDP stimulation --- p.84 / Chapter 3.2.8 --- In vivo effect of NOD1,2 ligands on IgE and chemokine production in serum and BALF in allergic asthmatic mice --- p.89 / Chapter 3.3 --- Discussion --- p.93 / Chapter Chapter 4: --- NOD2 and TLR2 Ligands Mediated Activation of Basophils Interacting with Human Dermal Fibroblasts in Atopic Dermatitis --- p.100 / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Results --- p.102 / Chapter 4.2.1 --- Cell surface expression of adhesion molecules ICAM-1 on human dermal fibroblasts activated by heat-killed Staphyloccocus aureus (HKSA) --- p.102 / Chapter 4.2.2 --- Induction of chemokines upon the interaction of basophils and human dermal fibroblasts stimulated by HKSA --- p.104 / Chapter 4.2.3 --- Expression of NOD2 and TLR2 protein --- p.107 / Chapter 4.2.4 --- Cell surface expression of adhesion molecule ICAM-1 on human dermal fibroblasts activated by MDP, PGN, LTA or Pam3CSK4 --- p.110 / Chapter 4.2.5 --- Induction of cytokine and chemokines upon the interaction of basophils (with or without IL-33 priming) and human dermal fibroblasts stimulated by MDP, PGN, LTA or Pam3CSK4 --- p.112 / Chapter 4.2.6 --- Direct interaction between human dermal fibroblasts and basophils was required for the release of IL-6, CXCL8, CCL2 and CCL5 upon the stimulation of MDP, PGN, LTA and Pam3CSK4 --- p.118 / Chapter 4.2.7 --- Effect of signaling molecular inhibitors on the expression of adhesion molecule ICAM-1 --- p.121 / Chapter 4.2.8 --- Effect of signaling molecule inhibitors on the release of cytokine and chemokines upon the stimulation by NOD2 and TLR2 ligands --- p.123 / Chapter 4.2.9 --- Differential activation of intracellular signaling pathways involved in the interaction of human dermal fibroblasts and basophilic KU812 upon stimulation of NOD2 and TLR2 ligands --- p.127 / Chapter 4.3 --- Discussion --- p.131 / Chapter Chapter 5: --- Effect of Tumor Necrosis Factor Family Member LIGHT on the Activation of Basophils Interacting with Bronchial Epithelial Cells: Potential Therapeutic Target for Airway Remodeling --- p.138 / Chapter 5.1 --- Introduction --- p.138 / Chapter 5.2 --- Results --- p.139 / Chapter 5.2.1 --- Cell surface expression of HVEM and LTβR --- p.139 / Chapter 5.2.2 --- Effect of LIGHT on the expression of ICAM-1 on basophil or BEAS-2B alone or co-culture --- p.141 / Chapter 5.2.3 --- Induction of cytokine and chemokine upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.144 / Chapter 5.2.4 --- Induction of MMP-9 upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.147 / Chapter 5.2.5 --- Effect of LIGHT on the release of TGFβ-1, histamine and periostin upon the interaction of basophils and BEAS-2B cells --- p.149 / Chapter 5.3 --- Discussion --- p.152 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.156 / Chapter 6.1 --- General conclusions --- p.156 / Chapter 6.2 --- Future perspectives --- p.160 / Appendix --- p.163 / References --- p.165

Results of the above studies thus suggested that immune dysregulation in SLE result in derangement of a spectrum of inflammatory mediators leading to possible multiple organs auto-inflammatory damages. However, the exact etio-pathogenic mechanism could not simply be explained by these phenomena. Infection has been invoked as an underlying etiology or trigger for the induction of autoimmune disease. Epstein-Barr virus (EBV) possesses multiple features that characterise its involvement in initiating or perpetuating SLE disease. Several research groups demonstrated that the peripheral blood EBV DNA load is significantly higher in SLE patients, yet cell-free viral DNA was also reported in other EBV-associated diseases such as nasopharyngeal carcinoma (NPC) and certain lymphomas, suggesting that relatively little is known about its biology and dynamic distribution in the blood circulation. In the second part of our study, we examined the cell-free and cell-associated distribution profile of EBV DNA load in SLE. Our data showed that the distribution of EBV DNA in the cell-free and cell-associated compartments exhibited a heterogeneous pattern in SLE patients. Contrary to the exclusive presence of circulating cell-free EBV DNA in NPC patients, both cell-free and cell-associated EBV DNA were detected in some SLE patients, while in others, no EBV DNA was measurable in either blood compartments. The level of cell-associated EBV viral load was significantly higher in SLE patients with active disease than those who presented with milder disease activity. This phenomenon indicated a possible association of EBV viral infection with the level of immune competence in SLE patients. It has been reported that EBV encodes proteins which shares significantly homology sequence with human IL-6, IL-8, IL-10, IL-12 and colony-stimulating factor (CSF)-1. This proposition brought our attention to the immune perturbation by EBV on the cytokine balance, possibly constitute in part, to the immune dysregulation and Th1 and Th2 dichotomy in SLE exacerbation. (Abstract shortened by UMI.) / The first section of this research study aimed to explore the messengers that influence Th1/Th2 cells differentiation, development, effector functions and hence their plausible contribution in SLE immunopathogenesis. We focused on studying the expression of cytokine and chemokine milieu that directs the traffic of T lymphocytes; co-stimulatory molecules in the activation of T lymphocytes; transcription factors T-bet and GATA-3 in regulating the differentiation of Th1 and Th2 cell lineage. We also investigated the involvement of the lymphocyte subpopulation, Th17 in the auto-inflammatory axis of SLE exacerbation. / Lit, Choi Wan. / Advisers: Christopher W.K. Lam; Y.M. Dennis Lo. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3358. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 203-235). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Chapter 1. Background: Asthma is the most common chronic childhood disease and is characterized by recurrent airway obstruction, bronchial hyper-responsiveness, and airway inflammation. Asthma is the leading cause of childhood hospitalization and school absenteeism in the United States. The associations between adverse respiratory effects and exposure to indoor nitrogen dioxide (NO2) and other byproducts of combustion such as particulate matter (PM) in particular ultrafine particulates (UFP), Ozone (O3) and Sulfur Dioxide (SO2), have been the focus of many epidemiological studies in recent years. Indoor exposure to NO2 and other pollutants from combustion may increase the risk of acute and chronic respiratory disease, reduce lung function, initiate and exacerbate asthma in children. The levels of exposure to NO2 indoors are of public health concern because children spend nearly 70% of their time indoors at home. According to the 2010 US Census report, approximately 39% of US households use natural gas for cooking, and the primary source of residential NO2 is a gas-fuel cooking appliance. Indoor levels of NO2 where NO2 sources are present can be much higher than outdoors, where the primary source of NO2 is vehicular traffic. Epidemiological studies in developed countries suggest that gas stoves used for cooking and/or heat are associated with an increased risk of asthma and respiratory symptoms in children. While there are numerous, epidemiological studies supporting an association between increased NO2 levels and gas stoves and asthma symptom severity in children, there are other studies that have examined the relationship in homes that did not observe significant associations. A better understanding of how NO2 and other indoor environmental (e.g., environmental tobacco smoke (ETS), allergens) exposures contribute to asthma morbidity in inner city preschool children will allow interventions to more effectively designed and implemented. To date, there are conflicting results on the role of exposure to indoor NO2 and its association with new-onset asthma in young inner-city children. The recent studies assessing the effects of indoor NO2 on asthma morbidity were limited to inner-city children, largely older, who were diagnosed with asthma. A gap in knowledge remains regarding the role indoor NO2 plays on the development of asthma in children not previously diagnosed. The scientific and public health rationale for conducting this dissertation was to describe the association of exposure to indoor NO2 and primary sources with the initiation and exacerbation of asthma symptoms among pre-school children with and without diagnosed asthma. The data analyzed in the current research come from a larger study of Endotoxin, Obesity, and Asthma (EOA) in the New York City Head Start Program, funded in the summer of 2002. The primary research objective of that study was to identify modifiable risk factors associated with asthma and asthma persistence among preschool children from low-income families living in select New York City neighborhoods with high pediatric asthma hospitalization rates. We conducted a cross-sectional analysis of data collected from the study questionnaire and home visit sampling at study enrollment. The analyses were performed in two phases: the first phases used data collected at study enrollment and the second phase used data collected 12-months after study baseline. Henceforth, the dissertation will refer to the first analyses as the baseline study and the second as the follow-up study. The research evaluated the association of NO2 exposure with asthma status among New York City Head Start children with and without asthma at study enrollment and with respiratory symptoms among children with asthma at 12-month follow-up. Chapter 2. Baseline Study: We conducted a cross-sectional analysis of data collected from the study questionnaire and home visit sampling at study enrollment. Specifically, the research sought to evaluate the association of NO2 exposure with asthma status among New York City Head Start children with and without asthma at study enrollment and with respiratory symptoms among children with asthma at enrollment. A total of 503 children were included in the baseline study. A total of 105 children (20.9%) met the criteria for both asthma and allergy, and 67 (13.3%) met the criteria for asthma alone. Girls made up 51.7% and boys, 48.3% of the 503 study participants. Descriptive analyses suggested that asthma/allergy status was associated with: male gender, non-Mexican ethnicity/national origin, presence of a smoker in the child’s home, number of smokers in the child’s home, self-reported parental history of asthma, mother’s education level and sensitization to one or more of the four allergens. Logistic regression models were used to investigate the magnitude and direction (as well as trend) of the association between childhood asthma and indoor NO2 sources in the child’s home. Chapter 3. Follow-up Study: Our follow-up study involved the analysis of the 12-month follow-up data from the study of Endotoxin, Obesity, and Asthma in the New York City Head Start Program funded in the summer of 2002. We focused on assessing the magnitude and direction of the associations of exposure to indoor NO2 levels (based on baseline NO2 measurements) with children’s asthma status and with symptom severity among asthmatics at 1-year follow-up. For the follow-up study, we categorized children by whether their asthma status had changed since baseline. Descriptive analyses were performed looking at key characteristics by “change in asthma status.” Children’s asthma status at baseline and at follow-up, were based on responses to the questionnaire. We analyzed indoor NO2 level measurements at baseline in relation to asthma outcomes on follow-up. We did not have enough data on NO2 levels at follow-up to analyze them in relation to asthma status on follow-up. Unless the family had relocated since baseline and/or reported changes since baseline in the use of gas appliances or the number of smokers in the home, we assumed that baseline NO2 levels in the participating children’s homes were reasonable proxies for current exposures. We looked at the number of children who moved since baseline and whether the move (for example, looking at gas stove status, age of new building) may have impacted indoor NO2 levels. Of the 503 children who were included in the baseline analyses, 47.3% had data on asthma status on follow-up. A total of 238 children (111 male, 127 female) were grouped into the four mutually exclusive outcome categories: 122 (51.3%) did not have asthma at baseline or on follow-up, 34 (14.3%) had asthma on follow-up but not at baseline, 65 (27.3%) had asthma at baseline but not on follow-up, and 17 (7.1%) had asthma at baseline and on follow-up. The mean age at 1-year follow-up was 59.5 months (6.95), and neither age nor gender was associated with asthma. The distribution of ethnicity/national origin among the 238 children remained the same as at baseline; no one ethnicity group experienced disproportionate loss to follow-up, and asthma status remained associated with non-Mexican ethnicity/national origin, although 44.1% with new-onset asthma were of Mexican background. Asthma was also associated with self-reported parental history of asthma and allergy in children, but nearly 80% of children with new-onset asthma had no such parental history of asthma. More parents of children with new-onset (35.3%) or persistent asthma (23.5%) than of other children reported making efforts to reduce risk factors or triggers for asthma exacerbations in the past 12 months. Chapter 4. Dissertation Conclusion : The primary objective of the dissertation research was the examination of the relationship between asthma and asthma severity and exposure to gas cooking and residential NO2. In both our baseline and 12-month follow-up studies, exposure to indoor NO2 was represented by the baseline measurement of NO2 and the NO2 surrogate, gas stove. Asthma status of children was based on parental responses on the questionnaire regarding asthma symptoms and urgent care visits due to respiratory distress over the course of each 12-month period prior to the conducting study questionnaires. For both studies, we did not find an association between exposure to NO2 levels at baseline and asthma status or severity. Our findings contradict the results of most recent studies of both NO2 levels and residential sources of NO2 and their effects on asthma symptoms in very young children. However, it remains difficult to compare our results we those of previous published studies because those studies primarily focused on children who were diagnosed with asthma, whereas our research included preschool aged children with and without asthma. Based on our findings and the fact they conflict with other epidemiological studies, of which there were also conflicting results, we feel that the relationship between asthma symptoms and NO2 exposures remains ambiguous. The lack of consistent results of epidemiological research raises questions that should be the focus of future epidemiological studies. What are the roles of co-pollutants and co-risk factors? Does NO2 work alone or in concert with other indoor pollutants? There exists a real lack of understanding on the possible synergistic effects of exposure to NO2 and other combustion byproducts. Important to furthering our knowledge of the role of exposure to indoor NO2 and asthma is determining whether NO2 acts as a surrogate for co-pollutants that are considered risk factors for asthma and other respiratory conditions. Another focus of future indoor pollution studies should be the development of effective methods and technologies for measuring the constituents of the complex mixture of pollutants in indoor air; these methods and technologies can then be applied in personal monitoring of exposure to indoor pollutants in epidemiological studies that would help to determine with much more accuracy the effects of individual indoor pollutants on asthma and other respiratory symptoms. This knowledge would help in the development of more effective public health and environment policies towards reducing the burden of childhood asthma.

A asma afeta aproximadamente 300 milhões de pessoas no mundo e é a maior causa de internação hospitalar em crianças nos países desenvolvidos. Essa doença é incurável e por vezes refratária ao tratamento em um número significativo de pacientes. As taxas de prevalência de tabagismo entre pacientes asmáticos são semelhantes aos da população em geral e o impacto da fumaça de cigarro nestes pacientes ainda é clinicamente controverso. O objetivo deste trabalho foi traçar o perfil inflamatório e analisar o papel das células dendríticas sobre a imunomodulação provocada pela exposição à fumaça do cigarro na inflamação alérgica pulmonar induzida previamente por ovalbumina (OVA) em um modelo murino. Primeiramente avaliamos in vivo a ação da fumaça de cigarro na inflamação pulmonar alérgica crônica avaliando a responsividade brônquica, o remodelamento pulmonar, a produção de anticorpos antígeno-específicos, o perfil de células inflamatórias pulmonares e sistêmicas e a produção de citocinas inflamatórias e moduladoras. Em seguida, realizamos estudo in vitro do perfil de maturação, migração e inflamatório de células dendríticas expostas a OVA e/ou a extrato de fumaça de cigarro. Nosso estudo mostrou que a sensibilização e desafios inalatórios com OVA levaram à inflamação pulmonar de característica Th2 com aumento de responsividade brônquica, remodelamento, altos níveis de IgE e de citocinas pró-inflamatórias como IL-4, IL-5 e IL-13. A exposição à fumaça de cigarro, surpreendentemente, levou a uma redução dos níveis de IL-4, IL-5 e IL-13, e simultaneamente reduziu os níveis de citocinas anti-inflamatórias como IL- 10 e TGF-beta em animais sensibilizados e desafiados com antígeno. Foi observada nestes animais, uma redução no número de eosinófilos no lavado broncoalveolar e aumento no número de neutrófilos no pulmão. A combinação da inflamação alérgica com exposição à fumaça de cigarro levou a um aumento do recrutamento e ativação de células dendríticas linfoides nos linfonodos mediastinais, que mostrou relação direta com aumento do influxo de células T CD8+ e ativação das mesmas no pulmão. A inflamação alérgica juntamente com a exposição à fumaça de cigarro, levou a uma redução no recrutamento de células dendríticas plasmocitoides além de reduzir o recrutamento de células T regulatórias. In vitro, mostramos que o extrato de fumaça de cigarro combinado ao antígeno aumenta a capacidade migratória e fagocítica do antígeno pelas BMDCs. No entanto, houve redução da expressão gênica para IL-13 neste mesmo grupo. Concluímos que neste modelo de inflamação pulmonar alérgica crônica combinada com a exposição à fumaça de cigarro leva a uma descaracterização do perfil inflamatório característico da resposta Th2 com a redução do recrutamento de eosinófilos, redução dos níveis de IL-4, IL-5 e IL-13 aliados a um aumento do número de neutrófilos, o que pode estar relacionado ao aumento do recrutamento e ativação de células dendríticas linfoides bem como de células T CD8+ e redução local de células dendríticas plasmocitoides. Mostramos ainda que a fumaça de cigarro juntamente com o antígeno leva as células dendríticas a aumentarem sua capacidade fagocítica porém, reduzir sua capacidade pró-inflamatória pela expressão gênica reduzida de IL-13 / Asthma affects approximately 300 million people worldwide and it is the major cause of hospitalization among children in developed countries. This disease is often refractory to treatment in a high number of patients. The prevalence rates of smoking among asthmatic patients are similar to the general population and the impact of cigarette smoke is also clinically controversial. The main goal of this study is to outline, in a murine model, the inflammatory profile and to analyze the role of dendritic cells on the immunomodulation caused by exposure to cigarette smoke in ovalbumin (OVA)-induced pulmonary allergic inflammation. First, we evaluated in vivo the action of cigarette smoke on chronic allergic pulmonary inflammation, evaluating the bronchial responsiveness, pulmonary remodeling, the production of antigen-specific antibodies, pulmonary and systemic inflammatory cell profile and the production of inflammatory and modulating cytokines. Next, we performed an in vitro study of the maturation, migration and inflammatory profile of dendritic cells exposed to OVA and/or cigarette smoke extract. Our study showed that sensitization and challenge with OVA led to Th2-type lung inflammation with increased bronchial responsiveness, remodeling, high levels of IgE and proinflammatory cytokines such as IL-4, IL-5 and IL-13. Exposure to cigarette smoke has surprisingly led to a reduction in levels of IL-4, IL-5 and IL-13, and simultaneously reduced levels of anti-inflammatory cytokines such as IL-10 and TGF-beta in animals sensitized and challenged with the antigen. We also observed a reduction in the number of eosinophils in bronchoalveolar lavage fluid and an increase in the number of neutrophils in the lung of these animals. The combination of allergic inflammation with exposure to cigarette smoke led to increased recruitment and activation of lymphoid dendritic cells in the mediastinal lymph nodes, which showed to be direct related with increased activation and influx of CD8+ T cells in lung. Allergic inflammation combined with cigarette smoke led to a decrease of plasmacytoid dendritic cells a well as regulatory T cells. In vitro, we showed that cigarette smoke extract combined with antigen increased migratory and phagocytic capacity of BMDCs. However, there was a reduction of IL-13 gene expression in this same group. We conclude that in this model of chronic pulmonary allergic inflammation combined with exposure to cigarette smoke leads to a mischaracterization of the characteristic inflammatory profile of the Th2 response with the reduction of eosinophil recruitment, reduction of levels of IL-4, IL-5 and IL-13 allied to increased number of neutrophils, which is related to increased recruitment and activation of lymphoid dendritic cells as well as CD8+ T cells and local decrement of plasmacytoid dendritic cells. We further show that cigarette smoke combined with antigen increases dendritic cell phagocytic capacity however, reduces its pro-inflammatory capacity by the reduced gene expression of IL-13

INTRODUÇÃO: Há evidências consistentes a partir de estudos epidemiológicos de que os profissionais de limpeza têm um risco elevado de desenvolver asma. Os determinantes deste risco não são totalmente conhecidos. Esses trabalhadores estão expostos a agentes ocupacionais de baixo e alto peso molecular, tanto a agentes sensibilizantes, como a irritantes. É importante produzir evidências de que este risco está relacionado ao trabalho e não às condições sociais ou outros fatores concorrentes, conhecer a anormalidade patológica subjacente, e investigar os possíveis agentes. O acúmulo deste conhecimento permitirá a proposição de medidas para substituição ou controle do uso dos agentes envolvidos e prevenção da ocorrência de novos casos desnecessariamente. Além disso, o uso de novas técnicas não invasivas, como a citologia do escarro e A FeNO, poderá facilitar o diagnóstico precoce dos casos. Desta maneira, este estudo pretende avaliar se o ambiente de trabalho induz inflamação pulmonar em trabalhadores assintomáticos, antes da alteração das provas funcionais e a eficácia do escarro induzido e da FeNO NO como marcadores de inflamação pulmonar precoce entre trabalhadores de limpeza profissional não doméstica. MÉTODO: Os trabalhadores foram avaliados através da comparação da citologia do escarro, valores da FeNO, espirometria e PFE, realizados durante o período de trabalho e após as férias. A amostra foi caracterizada através do questionário de triagem do estudo de saúde respiratória da Comunidade Européia, questionário de sintomas respiratórios e a pontuação no ISAAC. RESULTADOS: Em nosso estudo, encontramos um aumento significativo dos valores do VEF1 após o período de férias, (pré 2,76 ± 0,57 e pós 2,94 ± 0,61; p < 0,05) apesar de estar dentro da normalidade, em ambos os períodos. A média das medidas do PFE também mostrou-se maior durante o período de férias em comparação ao período de trabalho, embora não estatisticamente significante (pré 366,6 ± 54,1 e pós 386,4 ± 62,9 e p > 0,05). Encontramos uma redução dos valores da medida da FeNO após as férias (pré 16,3 ± 9,7 e pós 13,8 ± 7,8 p < 0,05) e redução de eosinófilos (pré 0,019 ± 0,05 e pós 0,003 ± 0,01 p < 0,05), linfócitos (pré 0,16 ± 0,35 e pós 0,01 ± 0,09 p < 0,05) e macrófagos (pré 0,421 ± 0,47 e pós 0,235 ± 0,30 p < 0,05) na citologia do escarro induzido, realizada após o período de férias. CONCLUSÃO: Demonstramos que o ambiente ocupacional ao qual são expostos os trabalhadores de limpeza profissional não doméstica provoca inflamação nas vias aéreas de trabalhadores assintomáticos. Esta inflamação pode ser aferida por métodos não invasivos como escarro induzido e FeNo, antes do aparecimento de alterações nas provas funcionais, embora estes métodos ainda necessitem de padronização. São necessários novos estudos para quantificar a exposição ao cloro e sua relação com inflamação, assim como para padronizar o uso do escarro induzido e da FeNO no diagnóstico de doenças ocupacionais entre trabalhadores de limpeza, além de medidas preventivas e educativas nesta população / There is consistent evidence from epidemiological studies that the cleaning professionals have a high risk of developing asthma. The determinants of this risk are not fully known. These workers are exposed to occupational agents of low and high molecular weight, both the sensitizing agents, such as irritant. It is important to produce evidence that this risk is related to work and not social conditions or other competitive factors, know the underlying pathological abnormality, and investigate possible agents. The accumulation of this knowledge will allow proposing measures to replace or control the use of the agents involved and preventing the occurrence of new cases unnecessarily. In addition, the use of new non-invasive techniques, such as sputum cytology and the FeNO may facilitate early diagnosis of cases. Thus, this study aims to assess if the work environment induces lung inflammation in asymptomatic workers, before the change of functional tests and the effectiveness of induced sputum and exhaled NO as early lung inflammation markers between professional cleaning workers. METHOD: Workers were evaluated by comparing the sputum cytology, FeNO values, spirometry and PEF, made during the work period and after the holidays. The sample was characterized by screening questionnaire of respiratory health study of the European Community, questionnaire of respiratory symptoms and a score in ISAAC. RESULTS: In our study, we found a significant increase in FEV1 values after the vacational period, (pre 2.76 ± 0.57 and 2.94 ± 0.61; post; p < 0.05) despite of being within the normal range in both periods. The average peak flow measurements also was higher during the vacational period compared to the period of work, although not statistically significant (366.6 ± 54.1 pre and post 386.4 ± 62.9; p > 0.05). We found a reduction of the exhaled measured values of NO after the holidays (pre and post 16.3 ± 9.7, 13.8 ± 7.8; p < 0.05), reduction of eosinophils (pre and post 0.05 ± 0.019, 0.003 ± 0.01; p < 0.05), lymphocytes (pre and post 0.16 ± 0.35, 0.01 ± 0.09; p < 0.05) and macrophages (pre and post 0.421 ± 0.47 0.235 ± 0 30 p < 0.05) in induced sputum cytology, performed after the holiday period. CONCLUSION: We demonstrate that the occupational environment to which professional cleaning non-domestic workers are exposed causes inflammation in the airways of asymptomatic workers. This inflammation can be measured by non-invasive methods such as induced sputum and FeNo, before the onset of changes in functional tests, although these methods still require standardization. Further studies are needed to quantify the exposure to chlorine and its relation to inflammation, as well as to standardize the use of induced sputum and exhaled nitric oxide in the diagnosis of occupational diseases among cleaning workers, and preventive and educational measures in this population

Kung, Wing Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 92-115). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.vii / Table of contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review --- p.4 / Chapter 2.1 --- Food allergy and its prevalence --- p.4 / Chapter 2.2 --- Mechanism and clinical symptoms of food allergy --- p.6 / Chapter 2.3 --- Tropomyosin as the major allergen in shellfish --- p.15 / Chapter 2.4 --- Cross reactivity and epitope mapping of tropomyosin --- p.21 / Chapter 2.5 --- Novel approaches for the treatment of food allergy --- p.29 / Chapter Chapter 3. --- Expression of shrimp recombinant tropomyosin and sensitization of mice --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials and Methods --- p.40 / Chapter 3.2.1 --- "Recovery of E, coli with tropomyosin-carrying plasmid" --- p.40 / Chapter 3.2.2 --- Preparation of tropomyosin-carrying plasmid --- p.41 / Chapter 3.2.3 --- Confirmation of DNA sequence of the tropomyosin --- p.41 / Chapter 3.2.4 --- Identification of the recombinant protein --- p.43 / Chapter 3.2.5 --- Purification of the recombinant protein --- p.43 / Chapter 3.2.6 --- Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 3.2.7 --- Concentration measurement of the recombinant tropomyosin --- p.45 / Chapter 3.2.8 --- Mice --- p.46 / Chapter 3.2.9 --- Mice sensitization and challenging --- p.46 / Chapter 3.2.10 --- Tropomyosin-specific IgE level in blood --- p.47 / Chapter 3.2.11 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.52 / Chapter 3.3.1 --- DNA sequence of the cloned tropomyosin --- p.52 / Chapter 3.3.2 --- Expression and purification of tropomyosin --- p.52 / Chapter 3.3.3 --- Hypersensitivity symptoms after challenge --- p.53 / Chapter 3.3.4 --- Blood tropomyosin-specific IgE level --- p.53 / Chapter 3.4 --- Discussion --- p.62 / Chapter Chapter 4. --- Identification of T cell epitopes --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Materials and methods --- p.67 / Chapter 4.2.1 --- Soluble epitope peptide synthesis --- p.68 / Chapter 4.2.2 --- Isolation of spleen cells from mice --- p.69 / Chapter 4.2.3 --- T cell proliferation assay --- p.70 / Chapter 4.3 --- Results --- p.71 / Chapter 4.3.1 --- Splenocyte proliferation to synthetic peptide --- p.72 / Chapter 4.3.2 --- Splenocyte proliferation to synthetic peptides pool --- p.72 / Chapter 4.4 --- Discussion --- p.77 / Chapter Chapter5 --- General conclusion --- p.89 / References --- p.92

Les travaux présentés dans cette thèse concernent le développement de nouveaux outils d'analyse multiparamétrique de type biopuce ou puce électrochimique, appliqués au diagnostic et au contrôle environnemental. Le premier axe de recherche a pour objectif le diagnostic, par la détection de panels d'anticorps marqueurs d'un état pathologique dans le sérum de patients. Dans cette optique, deux systèmes d'immunotests ont été développés, permettant la détection multiparamétrique d'anticorps spécifiques grâce à l'analyse automatisée et haut-débit d'échantillons de sérum. Cette approche s'est basée sur la capture des anticorps cibles par des sondes antigéniques immobilisées selon une matrice de plots sur des membranes constituant les fonds de puits de micro-plaques. La détection des interactions est effectuée par colorimétrie à l'aide d'un marqueur enzymatique. Ces outils ont permis l'analyse de 96 échantillons en moins de trois heures, et ont été mis au point pour deux applications. La première consiste en le diagnostic d'allergies, et la seconde s'intéresse au diagnostic du cancer. La seconde partie des travaux est appliquée au contrôle environnemental par surveillance de l'eau. Des pesticides, toxines et explosifs ont été définis comme composés cibles du test multiparamétrique. Afin de les intégrer dans une matrice de plots, des conjugués sondes ont été synthétisés à partir de ces haptènes. Après criblage et optimisation des conjugués en fonction de leur réactivité et réactivité-croisée avec les anticorps spécifiques, l'outil développé a démontré ses performances analytiques en termes de sensibilité et de sélectivité pour la détection des cibles. Un autre capteur pour la surveillance de l'eau a été développé dans le cadre du projet Européen BONAS. Ce test électrochimique vise à détecter des précurseurs d'explosifs utilisés dans la préparation de systèmes explosifs improvisés, pour la localisation de fabriques de bombes artisanales. La puce mise au point consiste en un réseau d'électrodes sérigraphiées, modifiées par électrodépôt de différents métaux / The work reported in this thesis focuses on the development of new multiplex analytical devices on biochip or electrode microarray format, dedicated to diagnosis and environmental monitoring. The objective of the first research axis is diagnosis, thanks to the detection in patients’ serum of a panel of antibodies, biomarkers of a pathological state. For that purpose, two immunotests have been developed, enabling the multiparametric detection of specific antibodies by automated and high-throughput analysis of serum samples. This approach is based on the antibodies capture by antigens probes immobilized in a matrix of spots on a membrane surface composing the wells bottom of a micro-titer plate. Enzyme-labeled antibodies have been used, providing a colorimetric detection. This device enabled the achievement of the analysis of 96 samples in less than three hours and has been applied to different applications. The first one consists of allergy diagnosis, and the second focuses on cancer diagnosis. The second part of this work is applied to environmental monitoring, through water analysis. Different types of pollutants have been defined as targets: pesticides, toxins and explosives. In order to integrate them in a matrix of probes, different conjugates have been synthesized with these haptens. After screening and optimization of the conjugates through their reactivity and cross-reactivity with the specific antibodies, the developed device demonstrated his analytical performances in terms of sensitivity and selectivity. Finally, for the European Project BONAS, a last sensor based on water analysis has also been developed. This electrochemical microarray aims to detect explosives precursors, used in improvised explosive devices, for the localization of hidden bomb factory. The chip was designed as a screen-printed electrode network, which was modified by different metals electrodeposition

<p>The eosinophil cationic protein (ECP) is usually associated with the eosinophil granulocyte. In this thesis the presence and production of this protein has been studied in two other cells. The circulating monocyte was found to contain ECP mRNA and small amounts of ECP, one thousand times less than that found in the eosinophil. The production decreased by differentiation of the myelomonoblastic cell line U937 into a macrophage phenotype. Submucosal lung macrophages did not stain for ECP and alveolar macrophages did not contain ECP mRNA. The circulating neutrophil contains ECP at a level hundred fold less than the eosinophil. We found that the protein is located to the primary granules of the neutrophil but could detect no ECP mRNA in the cell. It was shown in vitro that the protein was taken up by the cell and partly transported to the primary granules. The uptake did not seem to be receptor mediated. Upon stimulation of the neutrophils, ECP previously taken up, was re-secreted. </p><p>The ECP protein is heterogeneous both to molecular characteristics and to function. To evaluate if a genetic component is involved, the ECP gene was analysed in 70 individuals. Three single nucleotide polymorphisms (SNP´s) were found, denoted 277(C>T), 434(G>C) and 562(G>C). The two first were located to the mature peptide-coding region and would change the amino acids, arg45cys and arg97thr. The prevalence of the most common SNP, 434, was evaluated in two eosinophil-related diseases, allergy/asthma and Hodgkin Lymphoma (HL). Forty-three HL patients were evaluated and it was found that the 434GG was significantly more prevalent in patients having nodular sclerosis (NS) as compared to other histologies (p=0.03). Erythrocyte sedimentation rate was also related to the 434GG genotype (p=0.009). In 209 medical students 434GG was more common (p=0.002) in those who indicated allergy. The genotype was unrelated to the production of IgE antibodies to allergens. In analysis of 76 subjects with asthma it was found that the 434GG genotype was significantly more common among allergic asthmatics (p=0.04). Asthma and HL-NS are characterized by fibrosis and eosinophils and ECP has been suggested in fibrosis development. </p>

<p><i>Chlamydia pneumoniae</i> is a human respiratory tract pathogen. Seroepidemiological studies indicate that <i>C. pneumoniae</i> infection is most common in school-aged children and infrequently detected in younger children.</p><p>The aims of this study were to further elucidate the prevalence of <i>C. pneumoniae</i> in paediatric populations and to describe the clinical implications of these infections.</p><p>The study population consisted of 367 children with respiratory tract diseases, 453 presumed healthy children at day-care, 69 children undergoing adenoidectomy and 1585 children from a population based cohort. Family members to infected day-care children were investigated. The laboratory methods used were polymerase chain reaction (PCR) on specimen from upper respiratory tract, serology by microimmunofluorescence (MIF), and immunohistochemistry (IHC) on adenoid tissue specimen. Personal data and medical history were obtained by the means of questionnaires and by the study of patient records.</p><p>In children younger than five years, the prevalence of <i>C. pneumoniae</i> was 17% as detected by PCR. This prevalence started to increase with increasing age from two years of age. The corresponding increase in serology as detected by MIF started at the age of four years. The prevalence at day-care centres varied from 4 to 39%. Both PCR and MIF underestimated the prevalence of <i>C. pneumoniae</i> detected by IHC. Families to infected children were investigated: mothers were more often infected than fathers were.</p><p>Most <i>C. pneumoniae</i> infections in small children were confined to the upper respiratory tract. These infections were usually mild or asymptomatic. Symptomatic disease may be of prolonged nature. No subsequent illness after <i>C. pneumoniae</i> infection was detected at follow-up after four years. In general, no association between <i>C. pneumoniae</i> and asthma was found, but <i>C. pneumoniae</i> may be of importance for asthma in some susceptible individuals. Previous <i>C. pneumoniae</i> infection reduced the risk for later atopy.</p><p>In conclusion, <i>C. pneumoniae</i> is a common finding in small children and most often causes relatively mild disease. If the acquisition of this infection early in life will have any implications for future health remains to be investigated.</p>

<p>Immunoglobulin E (IgE) is an important mediator in atopic allergies. This thesis describes the development of a therapeutic vaccine against IgE and its effects in rats and dogs. The development of an assay to determine IgE levels in dogs, and the finding of a chromosome region in rats that affects IgE levels are also reported. </p><p>The vaccine is a chimeric molecule consisting of the constant domains Cε2, Cε3 and Cε4 from IgE. The target domain of the vaccine is the Cε3 domain in the recipient species, which is the domain directly involved in receptor binding, while the flanking regions, Cε2 and Cε4, are from a distantly related mammal. In rats, the vaccine induced an immune response against circulating IgE, which decreased IgE levels by 90% and substantially reduced their allergic symptoms. Further, the effects of adjuvants in rats and dogs were evaluated, and when co-administered with the vaccine certain adjuvants were shown to increase the immune response against IgE. Mineral-oils were the most potent adjuvants in inducing a response against IgE, but metabolizable oils spiked with immunostimulatory substances were also efficient. </p><p>It was also shown that the therapeutic vaccine could induce a decrease in IgE levels in adult dogs, even though their initial levels were exceptionally high compared with humans. The IgE levels in 76 dogs ranged between 1 and 41 μg/ml while humans normally have around 150 ng/ml. However, the high IgE levels did not correlate to any specific breed, nor did they distinguish between dogs that were diagnosed as healthy and those suffering from atopic eczema, autoimmunity or skin parasites. </p><p>Regulation of total IgE levels probably involves many genes. In the final phase of the study, one candidate locus known to be involved in arthritis susceptibility in rats was investigated, and was found also to affect IgE levels.</p>