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Almost 2 billion people world-wide are infected with parasitic helminths. These complex multicellular eukaryotic organisms are capable of establishing long-term infections even in the face of an intact immune response. Typically, in these settings regulatory components of the immune response, such as Foxp3+ T regulatory cells (Tregs), become dominant, limiting protective effector responses towards the parasite. Helminths are thought to have evolved mechanisms, including release of immunomodulatory molecules termed excretory-secretory products (ES), to sway the balance between the regulatory and effector arms of the immune response to favour their persistence. In this thesis both the development of a protective immune response toward, and the potential manipulation of the immune response by, the rodent gastrointestinal nematode Heligmosomoides polygyrus have been studied. Firstly, the effects of H. polygyrus ES (HES) on bone-marrow derived dendritic cells (DCs) were analysed. Although HES did not alter the phenotype of the DC it was found to be able to suppress the ability of the DC to respond to inflammatory stimuli. This activity was lost when HES was heat-inactivated (hiHES). After adoptive transfer, HES-pulsed DCs were able to induce a HESspecific T helper (Th)2-type response even if co-treated with an inflammatory stimulus. Th2-type responses are protective against H. polygyrus infection. Surprisingly, the ability of HES to generate a Th2-response in a co-treatment situation was not related to its anti-inflammatory properties; DCs co-treated with hiHES and an inflammatory stimulus were able to drive an equivalent Th2-response to HES in this situation. Next, making use of mouse strains with different susceptibility phenotypes to primary H. polygyrus infection, potential mechanisms of resistance were characterised. Development of granulomas in the gut wall were found to be associated with reduced worm burdens. Furthermore, in highly susceptible C57BL/6 mice, production of IL-23 was shown to be counter-regulatory to this process, as mice on the same background but deficient in this cytokine have increased numbers of granulomas and dramatically enhanced resistance. Susceptibility to H. polygyrus was also considered at the level of epigenetic regulation. A protein that binds specifically to methylated DNA, methyl-CpG binding domain protein (MBD)2, was found to affect the proportion of Foxp3+ Tregs within the CD4+ T cell population in vivo. Additionally, in vitro induction of Foxp3 in response to TGF-β was enhanced in MBD2-/- CD4+ T cells. MBD2-/- mice had a trend towards increased worm burdens when infected with H. polygyrus, suggesting that the difference in proportion of Tregs may limit generation of an effector response. Finally, the ability of HES to directly affect the regulatory arm of the immune response was focussed upon. It was found that HES was able to induce Foxp3 expression in naïve peripheral T cells, and that this was mediated by stimulation of the TGF-β pathway. The TGF-β mimic was of parasite origin as a pan-vertebrate TGF-β antibody was unable to block its effects but sera from H. polygyrus infected animals was competent to do this. Activity of this type was not limited to HES as ES from the ovine helminth Haemonchus contortus was found to have the same property. These data imply that some helminth parasites have evolved mechanisms to support generation of Foxp3+ Tregs, thus favouring the regulatory arm of the immune response and hence their own persistence.

The type of Atlantic salmon immune response to amoebic gill disease was investigated by analysis of cytokine genes possibly related to the TH1, TH17 and Treg pathways, which were significantly down regulated, while il-4/13 isoforms, possibly related to the TH2 pathway, were found to be significantly up regulated. Moreover, the injection of Atlantic salmon with rIfn-γ, which might initiate the TH1 immune pathway, did not reduce infection load of Paramoeba perurans or severity of gill pathology in challenged fish. Different arginase isoforms present in salmonids were also characterised and the data supported the concept that arginase type II may be a more relevant marker of alterantive activate macrophages in teleost fish induced by rIl-4/13. Regarding the biology of P. perurans, the susceptibility of amoebae to different environmental conditions showed that amoebae exposed to salinities lower than 3 ppt were disrupted or did not recover after 16 days, while all amoebae cultures showed a significant difference between the two temperatures (10°C and 15°C) studied over time. Significant differences were also found in relative abundance of the 30 most prevalent bacterial genera present in the isolated P. perurans cultures (16S rRNA). The impact of stress on the host response to AGD was tested for the presence of an association between temperature (10°C and 15°C) and variation in severity of AGD in Atlantic salmon. This association was demonstrated for the histopathology and P. perurans load analysis, reflecting an earlier and stronger AGD infection at the higher temperature (15°C) treatment. No significant difference between the two temperature treatments was shown in hormonal and molecular responses. Therefore, temperature might not act as a chronic stressor but its effect could be linked to the higher amoebae attachment seen at higher temperatures in the in vitro experiment reported.

Vaccination and the use of immune stimulants are two important ways to mitigate the costs of disease in fish aquaculture. A vaccine to Enteric redmouth disease (ERM) was the first licensed fish vaccine in the world. Although effective in protecting fish from the motile bacterial (Yersinia ruckeri) infection, ERM can occur in ERM vaccinated fish due to the rise of non-motile Y. ruckeri that does not express flagellin. This highlighted the need for continual improvement of vaccine efficacy and the importance of flagellin in fish immune responses. In this thesis the immune response to the ERM vaccine was studied first to give insights for vaccine development. A recombinant flagellin from Y. ruckeri (YRF) was then produced and its bioactivities were investigated in vitro and in vivo. The immune response to ERM vaccination was studied in rainbow trout in two major and relevant immune organs, the spleen and gills. Intraperitoneal injection of the ERM vaccine induces an early balanced expression of pro- and anti-inflammatory cytokines and adaptive cytokines in the spleen, with a heightened expression of acute phase proteins (APPs) and anti-microbial peptides (AMPs) in both spleen and gills. The analysis suggests that ERM vaccination activates host innate immunity and the expression of specific IL-12 and IL-23 isoforms leading to a Th1 and Th17 biased immune responses. This study has increased our understanding of the host immune response to ERM vaccination and the adaptive pathways involved. The early responses of a set of genes established in this study may prove useful as biomarkers in future vaccine development in aquaculture. YRF was next produced in a bacterial system, and purified. Its bioactivity was investigated first in the trout macrophage cell line RTS-11 and head kidney primary cell cultures. YRF is a potent activator of pro-inflammatory cytokines, APPs, AMPs and subunits of the IL-12 cytokine family in vitro. This property was further confirm in vivo in multiple tissues after intraperitoneal injection of YRF. These results suggest that flagellins are important pathogen-associated molecular patterns (PAMPs) that can activate an inflammatory response in fish not only in vitro but also in vivo. Furthermore, YRF was shown to be the most potent PAMP in vitro, in terms of activation of an inflammatory response, compared to pure LPS and peptidoglycan. In addition, YRF mixed with complete Freund's adjuvant can induce YRF-specific IgM antibodies in rainbow trout. These antibodies are able to neutralize YRF bioactivity, and react against the middle domain of YRF, as assessed in Western blot analysis. When YRF was fused with a protein antigen, it increased the antigen-specific IgM antibody response. This analysis reveals that YRF is a potent activator of host immune responses and can be used as an immune stimulant and adjuvant to improve vaccine efficacy.

Lee Ka Wai Sharon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 227-260). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABBREVIATIONS --- p.III / ABSTRACT --- p.VI / 摘要 --- p.XI / CONFERENCE PUBLICATIONS --- p.XVII / TABLE OF CONTENTS --- p.XVIII / Chapter Part I - --- General Introduction / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- The Search of Immunomodulatory Agents --- p.1 / Chapter 1.2. --- Cordyceps sinensis (Dong Cong Xia Cao) as an Immunomodulatory Agent --- p.2 / Chapter 1.2.1. --- General Aspects --- p.2 / Chapter 1.2.2. --- Evidence from the Traditional Chinese Medicine Concepts --- p.2 / Chapter 1.2.3. --- Evidence from the Traditional Chinese Medicine Classics --- p.4 / Chapter 1.2.4. --- Evidence from the Modern Research Literature --- p.4 / Chapter 1.2.4.1. --- lmmunomodulation --- p.4 / Chapter 1.2.4.2. --- Anti-tumor Effects --- p.7 / Chapter 1.2.4.3. --- Other Activities Related to the Immune System --- p.8 / Chapter 1.2.4.4. --- Potential Active Ingredients: Cordycepin and Polysaccharides --- p.8 / Chapter 1.2.5. --- Prescription and Usage: Single Vs Concocted --- p.11 / Chapter 1.2.5.1. --- Single Form as an Immunoactivating Agent --- p.11 / Chapter 1.2.5.2. --- Concocted as an Anti-asthmatic Agent --- p.12 / Chapter 1.3. --- Our Hypothesis and Rationale --- p.13 / Chapter Chapter 2: --- Experimental Design --- p.24 / Chapter 2.1. --- General Aspects of the Human Immune System --- p.24 / Chapter 2.2. --- Designing the In vitro Study on Cell-mediated Immunity --- p.24 / Chapter 2.2.1. --- T Cells --- p.25 / Chapter 2.2.2. --- Macrophages --- p.26 / Chapter 2.3. --- Designing the In vitro and In vivo Study of Anti-tumor Activities --- p.29 / Chapter 2.3.1. --- Tumor Biology --- p.29 / Chapter 2.3.2. --- Tumor and Immunity --- p.29 / Chapter 2.3.2.1. --- T-Cell-Mediated Cytolysis (Tc Cells) --- p.30 / Chapter 2.3.2.2. --- Delayed-Type Hypersensitivity (TDth Cells) --- p.30 / Chapter 2.3.2.3. --- Natural Killer (NK) Cells --- p.30 / Chapter 2.3.2.4. --- Lymphokine-Activated Killer (LAK) Cells --- p.31 / Chapter 2.3.2.5. --- Antibody-Dependent Cell-Mediated Cytotoxic (ADCC) Cells --- p.31 / Chapter 2.3.2.6. --- Activated Macrophages (AMΦ) --- p.31 / Chapter 2.3.3. --- Mechanism of Tumor Engulfment --- p.32 / Chapter 2.3.4. --- The Experimental Plan --- p.33 / Chapter 2.4. --- Designing the In vitro Study and Clinical Trials on Anti-asthmatic Activities --- p.36 / Chapter Part II - --- Methodology / Chapter Chapter 3: --- Materials and Methods / Chapter 3.1. --- List of Materials and Their Origin --- p.39 / Chapter 3.1.1. --- Traditional Chinese Medicine --- p.39 / Chapter 3.1.2. --- Cells for In vitro Experiments --- p.39 / Chapter 3.1.3. --- Mice for In vivo Experiments --- p.40 / Chapter 3.1.4. --- "Medium, Buffer, Supplements and Reagents for Cell Culture" --- p.40 / Chapter 3.1.5. --- Dye for Cellular Staining --- p.40 / Chapter 3.1.6. --- Cell Mitogens and Activator --- p.41 / Chapter 3.1.7. --- Reagents for Flow Cytometric Analysis --- p.41 / Chapter 3.1.8. --- Reagent Kits --- p.41 / Chapter 3.1.9. --- ELISA Kits --- p.42 / Chapter 3.1.10. --- Antibodies --- p.43 / Chapter 3.1.11. --- Reagents for RNA Extraction --- p.44 / Chapter 3.1.12. --- Reagents for Gel Electrophoresis --- p.44 / Chapter 3.1.13. --- Reagents for cDNA Expression Array --- p.44 / Chapter 3.1.14. --- Other Reagents --- p.45 / Chapter 3.1.15. --- Special Equipment and Apparatus --- p.45 / Chapter 3.2. --- Details of Materials --- p.46 / Chapter 3.2.1. --- Traditional Chinese Medicine --- p.46 / Chapter 3.2.1.1. --- Natural Cordyceps sinensis --- p.46 / Chapter 3.2.1.2. --- HERBSnSENSEŚёØ Cordyceps --- p.46 / Chapter 3.2.1.3. --- Wheeze-Relief Formula --- p.46 / Chapter 3.2.2. --- "Media, Supplements and Reagents for Cell Culture" --- p.47 / Chapter 3.2.2.1. --- Cell Culture Media --- p.47 / Chapter 3.2.2.2. --- Serum Supplements --- p.47 / Chapter 3.2.2.3. --- Anti-CD16 Magnetic Microbeads --- p.47 / Chapter 3.2.2.4. --- Fico´HёØ-Paque Plus Solution --- p.47 / Chapter 3.2.2.5. --- PercolĺёØ Solution --- p.48 / Chapter 3.2.2.6. --- Phosphate Buffered Saline (PBS) --- p.48 / Chapter 3.2.2.7. --- Water --- p.48 / Chapter 3.2.3. --- Dye for Cellular Staining --- p.48 / Chapter 3.2.3.1. --- HemacoloŕёØ for Microscopy --- p.48 / Chapter 3.2.3.2. --- Trypan Blue Dye --- p.49 / Chapter 3.2.4. --- Reagents for Flow Cytometry --- p.49 / Chapter 3.2.4.1. --- FACS Flow Sheath Fluid --- p.49 / Chapter 3.2.4.2. --- FACS Wash Medium --- p.49 / Chapter 3.2.4.3. --- Paraformaldehyde --- p.49 / Chapter 3.2.5. --- Special Equipments and Apparatus --- p.49 / Chapter 3.2.5.1. --- Magnetic Cell Sorting System (MACS) --- p.49 / Chapter 3.3. --- Human Subjects --- p.51 / Chapter 3.3.1. --- Inclusion Criteria --- p.51 / Chapter 3.3.2. --- Exclusion Crtieria --- p.51 / Chapter 3.3.3. --- Medication --- p.52 / Chapter 3.3.4. --- Informed Consent and Patient Information --- p.52 / Chapter 3.4. --- Animals --- p.53 / Chapter 3.4.1. --- Maintenance --- p.53 / Chapter 3.4.2. --- Survival Experiment Using Erhlich Ascites Tumor Bearing ICR Mice --- p.53 / Chapter 3.4.3. --- Experiments of Immunomodulatory activity in Sarcoma 180 Bearing BALB/c Mice --- p.54 / Chapter 3.5. --- Methodology --- p.55 / Chapter 3.5.1. --- Preparation of the Traditional Chinese Medicine --- p.55 / Chapter 3.5.1.1. --- Hot Water Extraction of Water Soluble Fraction of Natural Cordyceps sinensis --- p.55 / Chapter 3.5.1.2. --- Hot Water Extraction of Water Soluble Fraction of HERBSnSENSEŚёØ Corydceps and the Wheeze-relief Formula for In vitro Experiments --- p.55 / Chapter 3.5.1.3. --- HERBSnSENSEŚёØ Corydceps for the In Vivo Experiments --- p.56 / Chapter 3.5.1.4. --- Extraction Efficiency of the Hot Water Extracts --- p.56 / Chapter 3.5.2. --- Limulus Ameobocyte Lysate Test --- p.56 / Chapter 3.5.3. --- Cell Preparation --- p.57 / Chapter 3.5.3.1. --- "Isolation of Human Peripheral Blood Mononuclear Cells, Lymphocytes and Monocytes" --- p.57 / Chapter 3.5.3.2. --- Isolation of Eosinophils --- p.58 / Chapter 3.5.3.3. --- Isolation of Spleen Cells from BALB/c Mice --- p.58 / Chapter 3.5.3.4. --- "Murine Ehrlich Ascites Tumor (EAT), PU5-18, and Sarcoma 180 (SC-180) Cell Lines" --- p.59 / Chapter 3.5.3.5. --- Human Eosinophilic Leukemic Cell Line (EoL-1) --- p.59 / Chapter 3.5.3.6. --- Human Hepatocarcinoma Hep-3B Cell Line --- p.59 / Chapter 3.5.3.7. --- Human Leukemic Cell Line (HL-60) --- p.59 / Chapter 3.5.3.8. --- Human Mast Cell Line (HMC-1) --- p.60 / Chapter 3.5.4. --- Collection of Mouse Serum and Human Plasma --- p.60 / Chapter 3.5.5. --- Collection of Culture Supernatant --- p.60 / Chapter 3.5.6. --- The Trypan Blue Exclusion Assay --- p.61 / Chapter 3.5.7. --- Colorimetric 5-bromo-2'-deoxyuridine (BrdU) Cell Proliferation Enzyme Linked Immunosorbent Assay (ELISA) --- p.61 / Chapter 3.5.8. --- Immunophenotyping --- p.62 / Chapter 3.5.9. --- The Cytometric Bead Array (CBA) Kits --- p.62 / Chapter 3.5.10. --- Intracellular Florescence Staining for Reactive Oxygen Species --- p.63 / Chapter 3.5.11. --- The Intracellular Zymosan Florescence Assay --- p.64 / Chapter 3.5.12. --- Total Cellular RNA Extraction --- p.64 / Chapter 3.5.13. --- Gel Electrophoresis of RNA Integrity --- p.65 / Chapter 3.5.14. --- cDNA Expression Array --- p.65 / Chapter 3.5.15. --- Cell Staining Using Cytospin --- p.66 / Chapter 3.5.16. --- Annexin V-FITC/Propidium Iodide Apoptosis Detection --- p.66 / Chapter 3.5.17. --- Weighing the Spleen and Tumor --- p.67 / Chapter 3.5.18. --- Preparing Samples for the Eosinophilic Cationic Protein Fluoroenzymeimmunoassay --- p.67 / Chapter 3.5.19. --- Statistical Analysis --- p.67 / Chapter Part III - --- Results: Pre-functional Assays / Chapter Chapter 4: --- "Extraction, Endotoxin Measurement, In vitro Cytotoxicity Testing, and the Selection of Optimal Concentration" / Chapter 4.1. --- Extraction efficiency --- p.68 / Chapter 4.1.1. --- Introduction --- p.68 / Chapter 4.1.2. --- Results --- p.68 / Chapter 4.2. --- Endotoxin Level --- p.69 / Chapter 4.2.1. --- Introduction --- p.69 / Chapter 4.2.2. --- Results and Interpretation --- p.69 / Chapter 4.3. --- Cytotoxicity --- p.70 / Chapter 4.3.1. --- Introduction --- p.70 / Chapter 4.3.2. --- Results and Interpretation --- p.71 / Chapter 4.3.2.1. --- Peripheral Blood Mononuclear Cells (PBMC) --- p.71 / Chapter 4.3.2.2. --- Eosinophils --- p.72 / Chapter 4.4. --- The Optimal Concentration (OC) --- p.76 / Chapter 4.4.1. --- Introduction --- p.76 / Chapter 4.4.2. --- Results and Interpretation --- p.76 / Chapter Part IV- --- Results: Immunomodulatory Activities of Cordyceps sinensis as a Single Herb / Chapter Chapter 5: --- Mitogenic Activity --- p.80 / Chapter 5.1. --- Introduction --- p.80 / Chapter 5.2. --- Results --- p.80 / Chapter 5.3. --- Discussion --- p.81 / Chapter Chapter 6: --- Cytokines and Cytokine Receptors --- p.84 / Chapter 6.1. --- Introduction --- p.84 / Chapter 6.2. --- Results --- p.84 / Chapter 6.2.1. --- Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on the Induction of Cytokines from Lymphocytes --- p.84 / Chapter 6.2.1.1. --- TNFa --- p.84 / Chapter 6.2.1.2. --- IL-6 --- p.85 / Chapter 6.2.1.3. --- IL-10 --- p.85 / Chapter 6.2.2. --- Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on the Induction of Cytokines from Monocytes --- p.92 / Chapter 6.2.2.1. --- IL-1β --- p.92 / Chapter 6.2.2.2. --- IL-6 --- p.92 / Chapter 6.2.2.3. --- IL-10 --- p.97 / Chapter 6.2.2.4. --- TNFα --- p.97 / Chapter 6.2.3. --- Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on the Expression of Cytokine Receptor --- p.102 / Chapter 6.2.4. --- Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on the Gene Expression of Cytokines and Cytokine Receptors in Peripheral Blood Mononuclear Cells --- p.105 / Chapter 6.3. --- Discussion --- p.112 / Chapter Chapter 7: --- Macrophage Functions: Phagocytosis and Release of Reactive Oxygen Species (ROS) --- p.116 / Chapter 7.1 --- Introduction --- p.116 / Chapter 7.2. --- Results --- p.117 / Chapter 7.2.1. --- Phagocytosis --- p.117 / Chapter 7.2.2. --- Release of Reactive Oxygen Species (ROS) --- p.117 / Chapter 7.3. --- Discussion --- p.124 / Chapter Chapter 8: --- Apoptosis of Selected Cancer Cell Lines --- p.126 / Chapter 8.1. --- Introduction --- p.126 / Chapter 8.2. --- Results --- p.127 / Chapter 8.2.1. --- Differential Cytotoxic Effects of natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on Various Cancer Cell Lines In vitro --- p.127 / Chapter 8.2.2. --- Differential Anti-Proliferative Effects of natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on Various Cancer Cell Lines In vitro --- p.129 / Chapter 8.2.3. --- Differential Apoptotic Effects of Natural Cordyceps sinensis and HERBSnSENSEŚёØ Cordyceps on Various Cancer Cell Lines In vitro --- p.131 / Chapter 8.2.3.1. --- Peripheral Blood Mononuclear Cells --- p.131 / Chapter 8.2.3.2. --- Hepatocarcinoma Hep-3B --- p.131 / Chapter 8.2.3.3. --- Human Eosinophilic Leukemic Cell Line --- p.134 / Chapter 8.2.3.4. --- Human Mast Cell Line --- p.134 / Chapter 8.2.3.5. --- Human Leukemic Cell Line (HL-60) --- p.138 / Chapter 8.2.3.6. --- Murine Macrophages/Monocytes Cell Line PU5-18 --- p.138 / Chapter 8.2.3.7. --- Murine Erhlich Ascites Tumor (EAT) --- p.142 / Chapter 8.2.3.8. --- Murine Sarcoma 180 (SC-180) --- p.142 / Chapter 8.3. --- Discussion --- p.145 / Chapter Part V- --- Results: Immunomodulatory Activities of Cordyceps sinensis in Concoction / Chapter Chapter 9: --- The In vivo Animal Model --- p.147 / Chapter 9.1. --- introduction --- p.147 / Chapter 9.2. --- Results --- p.148 / Chapter 9.2.1. --- The ICR Mice Model --- p.148 / Chapter 9.2.1.1. --- In vivo Effects of Natural C. sinensis and HERBSnSENSEŚёØ Cordyceps on the Ascitic Fluid Production of ICR Mice --- p.148 / Chapter 9.2.1.2. --- Effects of Natural C. sinensis and HERBSnSENSEŚёØ Cordyceps on the Survival of Tumor-bearing ICR Mice --- p.149 / Chapter 9.3. --- The BALB/c Mice Model --- p.153 / Chapter 9.3.1. --- In vivo Effects of HERBSnSENSEŚёØ Cordyceps on Spleen and Tumor Weight --- p.153 / Chapter 9.3.2. --- Effects of HERBSnSENSEŚёØ Cordyceps on the Mitogenic Activities of Spleen Cells --- p.154 / Chapter 9.3.3. --- "In vivo Effects of HERBSnSENSEŚёØ Cordyceps on the Cell Surface Expression of CD3, CD4, and CD8" --- p.157 / Chapter 9.3.4. --- Effects of HERBSnSENSEŚёØ Cordyceps on the Cytokine Release from Cultured Spleen Cells --- p.161 / Chapter 9.3.4.1. --- TNFα --- p.161 / Chapter 9.3.4.2. --- IFNγ --- p.163 / Chapter 9.3.4.3. --- IL-2 --- p.163 / Chapter 9.3.4.4. --- IL-4 --- p.163 / Chapter 9.3.4.5. --- IL-6 --- p.167 / Chapter 9.3.4.6. --- IL-10 --- p.167 / Chapter 9.3.4.7. --- IL-12p70 --- p.167 / Chapter 9.3.4.8. --- Monocyte Chemoattractant Protein(MCP)-1 --- p.167 / Chapter 9.3.5. --- In vivo Effects of HERBSnSENSEŚёØ Cordyceps on the Cytokine Synthesis --- p.172 / Chapter 9.4. --- Discussion --- p.174 / Chapter Chapter 10: --- In vitro Studies on Eosinophils and Peripheral Blood Mononuclear Cells --- p.178 / Chapter 10.1. --- Introduction --- p.178 / Chapter 10.2. --- Results --- p.180 / Chapter 10.2.1. --- In vitro Effects of Wheeze-Relief Formula on the Survival of IL-5 Enhanced Eosinophils --- p.180 / Chapter 10.2.2. --- In vitro Effects of Wheeze-Relief Formula on the Degranulation of Eosinophils --- p.180 / Chapter 10.2.3. --- In vitro Effects of Wheeze-Relief Formula on the Surface Expression of Adhesion Molecules and Chemokine Receptors on Eosinophils --- p.183 / Chapter 10.2.4. --- In vitro Effects of Wheeze-Relief Formula on the Surface Expression of Adhesion Molecules on Eosinophils --- p.183 / Chapter 10.2.5. --- In vitro Effects of Wheeze-Relief Formula on the Cytokine Release from Peripheral Blood Mononuclear Cells --- p.187 / Chapter 10.2.6. --- In vitro Effects of Wheeze-Relief Formula on the Gene Expression Profile of Cytokines and Cytokine Receptors of Peripheral Blood Mononuclear Cells --- p.187 / Chapter 10.3. --- Discussion --- p.196 / Chapter Chapter 11: --- The Clinical Trial: Analysis of Serological Markers --- p.200 / Chapter 11.1. --- Introduction --- p.200 / Chapter 11.2. --- Results --- p.202 / Chapter 11.2.1. --- Demographic Data and Drop-out Cases --- p.202 / Chapter 11.2.2. --- Lung Function Test --- p.202 / Chapter 11.2.3. --- Steroid Dosage --- p.202 / Chapter 11.2.4. --- Serological Markers --- p.205 / Chapter 11.3. --- Discussion --- p.215 / Chapter Part VI - --- Conclusion / Chapter Chapter 12: --- Concluding Remarks and Future Perspectives --- p.217 / Chapter Part VII- --- Appendix / Parent Information Sheet --- p.222 / 家長資訊 --- p.223 / Consent Form --- p.224 / Licence to Conduct Animal Experiments --- p.225 / Bibliography --- p.227