No description available.

Trichuris trichiura is a gastrointestinal dwelling nematode that infects almost 500 million people worldwide. T. muris occurs naturally in mice and is very closely related the human whipworm, making it a suitable model to dissect the immune response against the parasite. Studies using the Trichuris muris system have identified CD4+ T cells as dictators of the outcome of infection. In wild type mice, infection with a high dose of T. muris eggs leads to resistance and worm expulsion, which are dependent on a Th2 response and the secretion of type 2 cytokines especially interleukin (IL) 13. Chronicity is dependent on a Th1 response and occurs when mice are infected with a low dose of T. muris eggs. It is well established that metabolic changes are essential to promoting T cell activation and effector function. Moreover, during chronic infection the host immune system is continuously exposed to parasite antigen, which represents a metabolic challenge. This thesis has investigated the importance of T cell metabolism during response against T. muris. Data presented here show that low and high dose T. muris infections promote upregulation of the glycolytic pathway in CD4+ T cells. During later stages of chronic infection, CD4+ T cells displayed supressed glycolysis and mitochondrial respiration, and may be due to metabolic modulation imposed by the parasite. Leucine uptake via the amino acid transporter Slc7a5 was previously shown to be required for mTORC1 activation and for T cell effector function. Data presented here show that in early stages following a high dose T. muris infection, mice that lack Slc7a5 in T cells have delayed worm expulsion, impaired production of antibodies, and lower levels of IL-13. Their CD4+ T cells present reduced glycolytic rates when compared to cells from cohoused infected wild type mice. However, at later stages of infection, antibody, IL-13 and glycolytic levels were restored together with worm expulsion. CD4+ T cells from the early stage of infection showed reduced phosphorylation of mTOR, which suggested that impairment of function was mTOR dependent. Indeed, mice lacking mTOR in T cells fail to expel a high dose of parasites. They showed abrogation of IL-13 production, impairment in antibody class switching and their CD4+ T cells failed to upregulate glycolysis. Thus, this thesis shows that mTOR is essential for the proper functioning of T cells during T. muris infection and efficient amino acid transport plays a significant role. Taken together, these data show that metabolic orchestration of T cell function influences the capacity to effectively control helminth infection and that even subtle changes in T cell metabolic control can have a major effect on response phenotype.

by Tsang Chi Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 137-155). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 撮要 --- p.ix / Publications --- p.xi / Table of contents --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Human mast cells and their pathological roles in inflammation --- p.1 / Chapter 1.1.1 --- Morphology of mast cells --- p.1 / Chapter 1.1.2 --- Mediators of mast cells --- p.1 / Chapter 1.1.3 --- Migration and activation --- p.3 / Chapter 1.1.4 --- Pathological roles of mast cells --- p.3 / Chapter 1.1.5 --- Human mast cell-1 (HMC-1) --- p.5 / Chapter 1.2 --- Cytokines as stimulator of mast cells in inflammation --- p.7 / Chapter 1.2.1 --- SCF --- p.7 / Chapter 1.2.2 --- TNF-α --- p.8 / Chapter 1.2.3 --- IL-13 --- p.8 / Chapter 1.2.4 --- IL-18 --- p.9 / Chapter 1.2.5 --- IL-25 --- p.9 / Chapter 1.3 --- Interaction of mast cells with inflammatory cells through adhesion molecules and chemokines --- p.11 / Chapter 1.3.1 --- Adhesion molecules on mast cells --- p.11 / Chapter 1.3.2 --- Chemokines released by mast cells --- p.12 / Chapter 1.4 --- Intracellular signaling pathways in mast cells --- p.16 / Chapter 1.4.1 --- p38-MAPK pathway --- p.16 / Chapter 1.4.2 --- ERK pathway --- p.17 / Chapter 1.4.3 --- NF-kB Pathway --- p.18 / Chapter 1.4.3 --- Cross-talking of pathways --- p.18 / Chapter 1.5 --- Signal transduction pathways and pharmacological intervention --- p.23 / Chapter 1.6 --- Traditional Chinese Medicine and pharmacological intervention --- p.25 / Chapter 1.6.1 --- Anti-allergic effects of traditional Chinese Medicine --- p.25 / Chapter 1.6.2 --- Anti-asthmatic effects of a newly developed Wheeze-Relief Formula --- p.26 / Chapter 1.7 --- Aims and scope of the study --- p.30 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- HMC-1 cell Line --- p.32 / Chapter 2.1.2 --- Media and reagents for cell culture --- p.32 / Chapter 2.1.3 --- Recombinant human cytokines --- p.33 / Chapter 2.1.4 --- "Signal transduction pathway inhibitors: PD98035, SB203580 and BAY 117082" --- p.34 / Chapter 2.1.5 --- Monoclonal antibodies and reagents for immunofluorescent staining --- p.34 / Chapter 2.1.6 --- Reagents and buffers for chemokine detection --- p.35 / Chapter 2.1.7 --- Reagents and buffers for total RNA extraction --- p.36 / Chapter 2.1.8 --- Reagents and buffers for reverse transcription 一 polymerase chain reaction (RT-PCR) --- p.37 / Chapter 2.1.9 --- Reagents and buffers for protein extraction --- p.40 / Chapter 2.1.10 --- Reagents and buffers for detection of activated signaling pathways --- p.41 / Chapter 2.1.11 --- Reagents and buffers for agarose gel electrophoresis --- p.42 / Chapter 2.1.12 --- Reagents and buffers for SDS-polyacrylamide gel electrophoresis (PAGE) --- p.43 / Chapter 2.1.13 --- Reagents and buffers for Western blot analysis --- p.45 / Chapter 2.1.14 --- Reagents and buffers for cDNA expression array analysis --- p.47 / Chapter 2.1.15 --- Reagents and buffers for cell viability and proliferation assay --- p.48 / Chapter 2.1.16 --- Reagent kit for endotoxin level assay --- p.49 / Chapter 2.2 --- Methods --- p.49 / Chapter 2.2.1 --- HMC-1 cell cultures --- p.49 / Chapter 2.2.2 --- Flow cytometry of cell surface expression of ICAM-1 and ICAM-3 --- p.50 / Chapter 2.2.3 --- Total cellular RNA extraction --- p.50 / Chapter 2.2.4 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.2.5 --- Agarose gel electrophoresis --- p.51 / Chapter 2.2.6 --- "Quantitative analysis of IL-8, IP-10,MCP-1 and RANTES" --- p.52 / Chapter 2.2.7 --- Quantitative analysis of 1-309 and MIP-1β --- p.52 / Chapter 2.2.8 --- Detection of phosphorylated-ERX and phosphorylated-p38 MAPK --- p.53 / Chapter 2.2.9 --- Detection of NF-kB activity --- p.53 / Chapter 2.2.10 --- Detection of phosphorylated-ATF-2 --- p.53 / Chapter 2.2.11 --- Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) --- p.54 / Chapter 2.2.12 --- Western blot analysis --- p.54 / Chapter 2.2.13 --- MTT assay --- p.55 / Chapter 2.2.14 --- Cell proliferation assay --- p.55 / Chapter 2.2.15 --- Hot water extraction of TCM --- p.56 / Chapter 2.2.16 --- Endotoxin level assay --- p.56 / Chapter 2.2.17 --- cDNA expression array analysis --- p.57 / Chapter 2.2.18 --- Statistical analysis --- p.57 / Chapter Chapter 3 --- Results / Chapter 3.1 --- The effects of cytokines on the expression of ICAM-1 and ICAM-3 on HMC-1 --- p.59 / Chapter 3.1.1. --- "SCF, TNF-α and IL-13 up-regulated ICAM-1 but not ICAM-3 expression on HMC-1 cells" --- p.59 / Chapter 3.1.2. --- "SCF, TNF-α and IL-13 up-regulated the mRNA expression of ICAM-1" --- p.59 / Chapter 3.1.3 --- "The combined treatment of SCF and TNF-α, and SCF and IL-13 showed synergistic and additive effect on ICAM-1 expression respectively" --- p.60 / Chapter 3.1.4 --- Synergistic up-regulation of ICAM-1 expression in combined treatment of SCF and TNF-α was dose-dependently enhanced by SCF --- p.60 / Chapter 3.2 --- "The effects of cytokines on the release of IL-8, IP-10, MCP-1, RANTES, 1-309 and MIP-1β from HMC-1 cells" --- p.66 / Chapter 3.2.1 --- "SCF induced the release of IL-8, MCP-1, RANTES, 1-309 and MIP-1β" --- p.66 / Chapter 3.2.2 --- "TNF-a induced the release of IL-8, IP-10, MCP-1, RANTES and 1-309" --- p.66 / Chapter 3.2.3 --- SCF and TNF-α did not enhance the proliferation rate of HMC-1 --- p.66 / Chapter 3.3 --- "The effect of SCF and TNF-α on the activation of ERK, p38 MAPK and NK-kB" --- p.71 / Chapter 3.3.1 --- SCF activated ERK but not p38 MAPK and NF-kB --- p.71 / Chapter 3.3.2 --- TNF-α activated p38 MAPK and NF-kB but not ERK --- p.71 / Chapter 3.4 --- The effect of inhibitors on the SCF and TNF-a-induced release of chemokines --- p.76 / Chapter 3.4.1 --- "The optimal dose of PD98059, SB203580 and BAY117082" --- p.76 / Chapter 3.4.2 --- "PD98059 suppressed the SCF induced IL-8, MCP-1, RANTES, 1-309 and MIP-1β release from HMC-1 cells" --- p.76 / Chapter 3.4.3 --- SB203580 and BAY117082 differentially suppressed the TNF-α induced chemokine release from HMC-1 cells --- p.77 / Chapter 3.5 --- The effect of inhibitors on the SCF and TNF-a-induced upregulation of ICAM-1 --- p.83 / Chapter 3.5.1 --- BAY117082 but not SB203580 suppressed the TNF-α-induced ICAM-1 expression --- p.83 / Chapter 3.5.2 --- PD98059 and BAY 117082 suppressed the combined treatment of SCF and TNF-α induced ICAM-1 expression --- p.83 / Chapter 3.6 --- "Effect of inhibitors on TNF-α and SCF-induced ERK, p38 MAPK and NF-kB activities in HMC-1 cells." --- p.85 / Chapter 3.6.1 --- PD98059 suppressed the SCF-induced activity of ERK --- p.85 / Chapter 3.6.2 --- SB203580 and BAY117082 suppressed the TNF-α induced p38 MAPKand NF-kB activity respectively --- p.85 / Chapter 3.6.3 --- PD98059 suppressed the enhanced NF-kB activity after the combined treatment of SCF and TNF-α for 18 hours --- p.86 / Chapter 3.7 --- Effect of TNF-α and SCF on the gene expression profile of inflammatory cytokines and receptors of HMC-1 cells. --- p.90 / Chapter 3.8 --- The effects of TCM on the SCF-induced 1-309 and MCP-1 from HMC-1 cells --- p.95 / Chapter 3.8.1 --- "Endotoxin level of Radix astragali, Radix Scutellariae, Radix stemonae, Bulbus Fritillariae cirrhosae and Cordyceps sinensis" --- p.95 / Chapter 3.8.2 --- The effects of TCM on the proliferation rate of HMC-1 cells --- p.95 / Chapter 3.9.3 --- The effects of TCM on the SCF-induced release of 1-309 from HMC-1 cells --- p.96 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Involvement of adhesion molecules and chemokines in mast cell-mediated immunological events --- p.107 / Chapter 4.2 --- HMC-1 as the in vitro mast cell model adapted in my project --- p.108 / Chapter 4.3 --- The effect of cytokines on the expression of ICAM-1 and ICAM-3 in HMC-1 cells --- p.109 / Chapter 4.4 --- The effect of cytokines on the release of chemokines in HMC-1 cells --- p.111 / Chapter 4.5 --- "The regulation of ICAM-1, IL-8, IP-10, MCP-1, RANTES, 1-309 and MIP-1β through p-38 MAPK, ERK and NF-kB signaling pathways in HMC-1 cells" --- p.115 / Chapter 4.6 --- Further characterization of HMC-1 cells using cDNA array --- p.119 / Chapter 4.7 --- Investigating the in vitro anti-allergic activities of a newly developed Wheeze-relief formula using cytokine-activated HMC-1 cells --- p.128 / Chapter 4.8 --- Concluding remarks and future prospective --- p.132 / References --- p.137 / Appendix --- p.156

Ng Sin Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-135). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 撮要 --- p.ix / Publications --- p.xi / Table of contents --- p.xiii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Allergic Diseases and Allergic Inflammation --- p.1 / Chapter 1.1.1 --- Prevalence of Allergic Diseases --- p.1 / Chapter 1.1.2 --- Common Allergic Diseases: Allergic Asthma --- p.1 / Chapter 1.1.3 --- Common Allergic Diseases: Atopic Dermatitis --- p.2 / Chapter 1.1.4 --- Allergic Inflammation --- p.3 / Chapter 1.2 --- The Inflammatory Leukocytes: Mast Cells and Eosinophils --- p.6 / Chapter 1.2.1 --- Characteristics of Mast Cells --- p.6 / Chapter 1.2.2 --- Mast Cells Distribution --- p.8 / Chapter 1.2.3 --- Mast Cells Subtypes --- p.8 / Chapter 1.2.4 --- HMC-1 Cells --- p.9 / Chapter 1.2.5 --- Characteristics of Eosinophils --- p.12 / Chapter 1.3 --- Adhesion Molecules in Allergic Diseases --- p.15 / Chapter 1.3.1 --- Adhesion Molecules and Leukocyte Migration --- p.15 / Chapter 1.3.2 --- Selectin --- p.17 / Chapter 1.3.3 --- Intermolecular Adhesion Molecules --- p.17 / Chapter 1.3.4 --- Integrin --- p.18 / Chapter 1.4 --- Cytokines and Chemokines in Allergic Diseases --- p.18 / Chapter 1.4.1 --- IL-6 --- p.20 / Chapter 1.4.2 --- CXCL1 --- p.21 / Chapter 1.4.3 --- CXCL8 --- p.21 / Chapter 1.4.3 --- CCL2 --- p.22 / Chapter 1.5 --- Intercellular Signal Transduction Pathways in Inflammation --- p.24 / Chapter 1.5.1 --- RAS-RAF-mitogen-activated Protein Kinases --- p.24 / Chapter 1.5.2 --- Janus Kinase/ Signal Transducers and Activators of Transcriptions Pathway --- p.27 / Chapter 1.5.3 --- Nuclear Factor-KB Pathway --- p.29 / Chapter 1.5.4 --- Phosphoinositide 3-Kinase Pathway --- p.31 / Chapter 1.6 --- Aims and Scope of the Study --- p.33 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- HMC-1 Cell Line --- p.35 / Chapter 2.1.2 --- Human Buffer Coat --- p.35 / Chapter 2.1.3 --- Human Mast Cell Chymase and TLR ligands --- p.35 / Chapter 2.1.4 --- Media and Reagents for Cell Culture --- p.36 / Chapter 2.1.5 --- Reagents and Buffers for Purification of Human Eosinophils --- p.37 / Chapter 2.1.6 --- Reagents and Buffers for Flow Cytmetry --- p.38 / Chapter 2.1.7 --- Reagents and Buffers for Total RNA Extraction --- p.41 / Chapter 2.1.8 --- Reagents and Buffers for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.42 / Chapter 2.1.9 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.45 / Chapter 2.1.10 --- Reagents and Buffers for Sodium Dodecyl Sulfate -polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.1.11 --- Reagents and Buffers for Western Blot Analysis --- p.48 / Chapter 2.1.12 --- Chemotactic Migration --- p.51 / Chapter 2.1.13 --- Signaling Transduction Inhibitors and Protein Synthesis Inhibitors --- p.51 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- HMC-1 Cell Cultures --- p.52 / Chapter 2.2.2 --- Purification of Buffy Coat Eosinophils by MACS and Eosinophil Culture --- p.52 / Chapter 2.2.3 --- Total Cellular RNA Extraction --- p.53 / Chapter 2.2.4 --- RT-PCR --- p.54 / Chapter 2.2.5 --- Agarose Gel Electrophoresis --- p.55 / Chapter 2.2.6 --- Flow Cytometry Analysis --- p.55 / Chapter 2.2.7 --- Protein Array Analysis of Cytokine Release --- p.57 / Chapter 2.2.8 --- Quantitative Analysis ofCXCLl --- p.58 / Chapter 2.2.9 --- Total Protein Extraction --- p.58 / Chapter 2.2.10 --- SDS-PAGE --- p.58 / Chapter 2.2.11 --- Western Blot Analysis --- p.59 / Chapter 2.2.12 --- Chemotactic Migration Analysis --- p.60 / Chapter 2.2.13 --- Statistical Analysis --- p.60 / Chapter Chapter 3 --- Effects of Mast Cell Derived Chymase on Human Eosinophils and the Signaling Mechanisms: Implication in Allergic Inflammation / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Results --- p.65 / Chapter 3.2.1 --- Effects of Chymase on Eosinophil Survival --- p.65 / Chapter 3.2.2 --- Effects of Chymase on the Adhesion Molecule Expression of Eosinophils --- p.68 / Chapter 3.2.3 --- Effects of Chymase on the Chemokinetic Properties on Eosinophils --- p.71 / Chapter 3.2.4 --- Effects of Chymase on the Release of Chemokines and IL-6 from Eosinophils --- p.73 / Chapter 3.2.5 --- Signal Transduction Mechanism Involved in Regulating Chymase-induced Effects on Eosinophils --- p.78 / Chapter 3.3 --- Discussion --- p.71 / Chapter Chapter 4 --- TLR-mediated Effects and Signal Transduction Mechanism of HMC-1 Cells / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Results --- p.97 / Chapter 4.2.1 --- Expression of Adhesion Molecules on HMC-1 Cells --- p.95 / Chapter 4.2.2 --- TLR Expression Profile on HMC-1 Cells --- p.97 / Chapter 4.2.3 --- Effects of TLR ligands on HMC-1 Cell Adhesion Molecule Expressions --- p.99 / Chapter 4.2.4 --- TLR7-induced Phosphorylation of ERK and Effects of PD98059 on TLR7-induced ERK Phosphorylation --- p.104 / Chapter 4.2.5 --- Effect of TLR7 Ligand on HMC-1 Cells Cytokine Release --- p.108 / Chapter 4.3 --- Discussion --- p.110 / Chapter Chapter 5 --- Conclusions and Future Perspectives / Chapter 5.1 --- Conclusions --- p.115 / Chapter 5.2 --- Future Perspectives --- p.117 / References --- p.120 / Appendix --- p.136

Ng, Sin Man. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 96-104). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

CD4+-T-Helferzellen sind in großer Zahl in der entzündeten Synovialmembran bei rheumatoider Arthritis (RA) sowie in Arthritismodellen vorhanden und spielen mit großer Wahrscheinlichkeit eine bedeutende Rolle in der Pathogenese von Arthritiden. Bei der präventiven Behandlung mit drei verschiedenen Anti-CD4 monoklonalen Antikörpern (mAk) im Modell der Adjuvansarthritis der Ratte (AA) wurden abhängig von dem jeweils eingesetzten mAk unterschiedliche klinische Verbesserungen beobachtet. Im Mittelpunkt der Untersuchungen stand deshalb die Suche nach Parallelen zwischen der unterschiedlichen klinischen Effizienz der Anti-CD4 mAk W3/25, OX35 (klinisch effizient) und RIB5/2 (klinisch ineffizient) bei der präventiven Therapie der AA und ihren in vitro Effekten auf TZell-Funktionen als Erklärung für die unterschiedlichen Therapieeffekte. Keine klaren Parallelen zur differentiellen klinischen Effizienz ergaben sich bei den folgenden Untersuchungen: 1.) Bestimmung der Affinitäten der mAk zum CD4-Molekül. 2.) Inhibition der Proliferation in der primären gemischten Lymphozytenkultur (1° MLC) mit CD4+-T-Zellen und CD8+-T-Zellen durch die drei mAk 3.) Beeinflussung der Produktion der Zytokine IL-2, IFNg, IL-10 und IL-4 in verschiedenen experimentellen Ansätzen (sekundäre MLC nach Anwesenheit der mAk in der 1° MLC, Kreuzvernetzung des CD4-Moleküls mittels der mAk nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR). 4.) Einfluss der drei Anti-CD4 mAk auf die TZR-vermittelte Apoptose. 5.) Mobilisierung von intrazellulärem Kalzium durch CD4-Kreuzvernetzung mittels der mAk. 6.) Aktivität der Tyrosinkinasen p56lck und p59fyn nach CD4-Kreuzvernetzung mittels der mAk. 7) Phosphorylierung des Shc-Adaptermoleküls durch CD4-Kreuzvernetzung mittels der drei mAk. 8.) Effekte der drei mAk auf die Aktivität der Transkriptionsfaktoren NF-AT und AP-1. Dagegen ergaben sich bei den Untersuchungen zur Produktion von TNFa und zur Aktivität des Transkriptionsfaktors NF-kB eindeutige Parallelen zur differentiellen klinischen Effizienz: 1.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR induzierte eine signifikant höhere Sekretion von TNFa als mit den mAk W3/25 und OX35. 2.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 vor einer Stimulation von CD4+-T-Zellen über den TZR führte zu einer signifikant stärkeren Erhöhung der Aktivität von NF-kB als mit den mAk W3/25 und OX35. Beide differentiellen Effekte könnten daher die Erklärung für die unterschiedliche klinische Effizienz der drei Anti-CD4 mAk darstellen.

Regulation of the immune response is mediated, in part, by the action of suppressor T cells (Ts). One intriguing aspect of these cells is the description of T cell suppressor factor (TsF): a soluble analog of the cell that shares many of its properties, such as the ability to bind free antigen (Ag) and suppress an Ag-specific immune response. The exact molecular nature of TsF and the relationship of TsF to Ts are unknown. The immune response to the small, bacterial protein, ferredoxin (Fd), was used as a model system to study TsF. A Fd-specific suppressor cell network has been described in mice that are genetically nonresponsive to this Ag. Previously, a soluble mediator, known as Fd11F, was found in the culture supernatant (SN) of the Ts hybridoma, Fd11. Fd11F possessed both Ag-binding activity and the ability to suppress the anti-Fd Ab response in mice. The TsF-specific monoclonal antibody, B16G, was used for both the recovery of Fd11F-enriched material from SN and its detection by the enzyme-linked immunosorbent assay. ' Further immunochemical, biological, and biochemical characterization of Fd11F was done with emphasis on describing the Ag-binding properties of Fd11F. It was found that Fd11F bound to solid- and liquid-phase Fd, and demonstrated preferential binding to the carrier determinant of the Ag. A spleen cell culture assay was devised which showed that Fd11F suppressed Ab production in a concentration-dependent manner. Additional experiments suggested that the suppressive effect was Ag-specific. The identification of the Ag-binding molecule was attempted by the fractionation of Fd11F-enriched material using high performance gel filtration or preparative SDS-PAGE (run under non-reducing conditions). Using SDS-PAGE, a unique, single polypeptide of about 30k relative molecular mass (Mr) was identified as the Ag-binding moiety of Fd11F. The possible relationship of this moiety to other identified materials is discussed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Human B lymphocytes are notoriously difficult to culture. Two to three days after plating, a sharp decline in viability and cell number can be observed. The objective of this study was to evaluate the effect of support cells on B cell viability in an in vitro human immune system construct. B cells were combined with dendritic cells (DCs) and cultured for various periods of time in the presence of one of three types of support cells: EA cells, HS-5 cells, and HS-27 A cells. The B cells were either in physical contact with the support cells, or allowed to interact through soluble factors in the media in order to determine if the effect on viability was contact dependent or independent. Viability was assessed using flow cytometry. Finally, two functional assays were performed to evaluate the ability of the cultured B cells to respond to an immune challenge. Both recall and nai've antigens were used. The B lymphocytes were then assessed for viability, proliferation and activation using flow cytometry. ELISPOT was also employed to determine if any antigen specific antibodies were produced by the B cells. It was found that while the support cells did improve viability, they did not produce consistent or reliable results. Additionally, B lymphocytes cultured in the presence of support cells or support cell conditioned media had no antigen specific tetanus response and reduced proliferation. Therefore, even though the support cells did under some conditions enhance lymphocyte viability, the lack of a positive functional response negates the value of using them in an experimental system.

BACKGROUND: Regulatory T cells (Tregs) are an essential subset of T cells that despite over 10 years of research have yet to be fully characterized. These suppressive immune cells, derived from the thymus, express high levels of interleukin-2 receptor alpha-chain (CD25). Tregs are needed to maintain self-tolerance and to control responses to non-self-antigen. The mechanism of Treg repression is unknown. The direct cell-to-cell contact through binding of cell surface molecules as well as secretion of suppressive cytokines has been shown to suppress the proliferation of Thl and Th2 cells against auto, allo and foreign antigens. The role of Tregs in regulating B cell response is also uncertain. The objective of this study is to determine how the removal of T regulatory cells can increase B cell responses in vitro. METHOD: This study focuses on the effect Tregs have on the generation of an antigen specific immune response in vitro. T cells with and without Tregs were co-cultured with monocyte derived dendrtic cells. The antigen specific activation was determined by analyzing cytokine production using intracellular cytokine staining, a flow based assay. Next, the effects of Tregs on both recall and naive B cell responses was analyzed using a co-culture of B cells, CD4 T cells with and without Tregs and monocyte-derived dendritic cells. Analysis of lymphoproliferation, activation, and antibody production was analyzed by using flow cytometry, Elispot and ELISA assays. RESULTS: An antigen specific response against gp120 was generated in naive T cell culture. Tregs were shown to inhibit antigen specific cytokine production in CD4 T cell culture to de novo antigens. The activation in the absence of Tregs was superior to the addition of exogenous factors of IL-2 and IL-7 with a third less non-specific background activation. When analyzed in a TT recall B cell assay, however, the removal of Tregs proved to have an inhibitory effect on antigen secreting cells detected by Elispot. This inhibition appeared at both a 1: 1 and 1 :4 T to B cell ratios though was slightly decreased at the 1 :4 ratio. The same was true for na1ve B cell assay showing a decrease in the generation ofMSPl-42 IgM antigen secreting cells. ELISA assays also confirmed the results showing a nearly 2.5 fold decrease in the amount of MSP 1-42 specific IgM Ab in the L TE cell culture supernatant. Conclusion: While the removal of T regulatory cells is beneficial for the activation of na1ve T cells, the removal of Tregs seems to be inhibitory to B cell activation in the LTE. This inhibitory effect maybe due to T cells becoming too stimulatory before culture with B cells. Studies involving a wider range of T to B cell ratios and culture times may be beneficial to determine if the depletion of Tregs would benefit this culture method.

CD4+-T-Helferzellen sind in großer Zahl in der entzündeten Synovialmembran bei rheumatoider Arthritis (RA) sowie in Arthritismodellen vorhanden und spielen mit großer Wahrscheinlichkeit eine bedeutende Rolle in der Pathogenese von Arthritiden. Bei der präventiven Behandlung mit drei verschiedenen Anti-CD4 monoklonalen Antikörpern (mAk) im Modell der Adjuvansarthritis der Ratte (AA) wurden abhängig von dem jeweils eingesetzten mAk unterschiedliche klinische Verbesserungen beobachtet. Im Mittelpunkt der Untersuchungen stand deshalb die Suche nach Parallelen zwischen der unterschiedlichen klinischen Effizienz der Anti-CD4 mAk W3/25, OX35 (klinisch effizient) und RIB5/2 (klinisch ineffizient) bei der präventiven Therapie der AA und ihren in vitro Effekten auf TZell-Funktionen als Erklärung für die unterschiedlichen Therapieeffekte. Keine klaren Parallelen zur differentiellen klinischen Effizienz ergaben sich bei den folgenden Untersuchungen: 1.) Bestimmung der Affinitäten der mAk zum CD4-Molekül. 2.) Inhibition der Proliferation in der primären gemischten Lymphozytenkultur (1° MLC) mit CD4+-T-Zellen und CD8+-T-Zellen durch die drei mAk 3.) Beeinflussung der Produktion der Zytokine IL-2, IFNg, IL-10 und IL-4 in verschiedenen experimentellen Ansätzen (sekundäre MLC nach Anwesenheit der mAk in der 1° MLC, Kreuzvernetzung des CD4-Moleküls mittels der mAk nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR). 4.) Einfluss der drei Anti-CD4 mAk auf die TZR-vermittelte Apoptose. 5.) Mobilisierung von intrazellulärem Kalzium durch CD4-Kreuzvernetzung mittels der mAk. 6.) Aktivität der Tyrosinkinasen p56lck und p59fyn nach CD4-Kreuzvernetzung mittels der mAk. 7) Phosphorylierung des Shc-Adaptermoleküls durch CD4-Kreuzvernetzung mittels der drei mAk. 8.) Effekte der drei mAk auf die Aktivität der Transkriptionsfaktoren NF-AT und AP-1. Dagegen ergaben sich bei den Untersuchungen zur Produktion von TNFa und zur Aktivität des Transkriptionsfaktors NF-kB eindeutige Parallelen zur differentiellen klinischen Effizienz: 1.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 nach bzw. vor einer Stimulation von CD4+-T-Zellen über den TZR induzierte eine signifikant höhere Sekretion von TNFa als mit den mAk W3/25 und OX35. 2.) Die Kreuzvernetzung des CD4-Moleküls mittels des mAk RIB5/2 vor einer Stimulation von CD4+-T-Zellen über den TZR führte zu einer signifikant stärkeren Erhöhung der Aktivität von NF-kB als mit den mAk W3/25 und OX35. Beide differentiellen Effekte könnten daher die Erklärung für die unterschiedliche klinische Effizienz der drei Anti-CD4 mAk darstellen.