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title page
Contents
I. Introduction 8
II. Patients and methods 12
1. Patients Selection 12
2. Preparation of Tumor Lysate 13
3. Preparation of DC 14
4. Phenotypic Analysis 17
5. Treatment Protocol 17
1) Immunotherapy protocol 17
2) Dose escalating schedule 19
6. Delayed Type Hypersensitivity 21
7. KLH-induced T-cell Proliferation Assay 21
8. Analysis of IFN-γ-producing Cells using ELISpot Assay 22
9. Clinical Response and Toxicity Assessment 23
III. Results 24
1. Patients Characteristics 24
2. Characteristics of Cultured DC 26
3. Dose of administered TL-pulsed DC 30
4. Clinical Outcome 30
5. Toxicity 35
6. DTH Reactivity 37
7. KLH-induced T-cell Proliferation 39
8/9. ELISpot Results for IFN-γ-producing Cells 41
IV. Discussion 43
V. Acknowledgements 50
VI. References 51
ABSTRACT 59
국문초록 62
Table 1. Dose escalation schedule 20
Table 2. Patient characteristics before vaccination 25
Table 3. The phenotypes of tumor lysate-pulsed dendritic cells generated in seven patients. HLA class II- and CD86-positive cells were about 90% of total cells and more than 95% of DC gated cells.... 29
Table 4. Details of vaccination and response to immunotherapy 32
Table 5. Toxicity profile by patients 36
Figure 1. Preparation of tumor lysate-pulsed dendritic cells 16
Figure 2. Immunotherapy protocol 20
Figure 3. Morphology of tumor lysate-pulsed dendritic cells derived from patient RCC-A-02. Representative images were obtained by confocal microscopy with a magnification of x200 (A) and x400 (B) at day 7.... 27
Figure 4. Phenotypic analyses of tumor lysate-pulsed dendritic cells (TL-pulsed DC) in patient RCC-A-03. Flow cytometry was performed using FACS caliber apparatus.... 28
Figure 5. Tumor regression after vaccination with tumor lysate-pulsed dendritic cells in patient RCC-B-04. CT scans before (A) and after (B) the first cycle of vaccination showed a marked regression or disappearance of metastatic... 33
Figure 6. Overall survival. With a median F/U of 13 months, the median OS has not been reached (range, 8 to 30 months). 34
Figure 7. Delayed type hypersensitivity (DTH) in RCC-A-03 and RCC-B-04 patient. Patients did not show DTH response to either KLH- or tumor lysate-pulsed dendritic cells (TL-pulsed DC) before vaccination.... 38
Figure 8. KLH-induced T-cell proliferation assay. PBL were seeded in flat-bottomed 96-well plates (1-2 × 105 cells/well) with KLH (0, 10, 50, 100 ㎍/ml) for 5 days, and loaded with 1μCi ³H-thymidine/well during the last 18 h....=(이미지참조) 40
Figure 9. ELISpot assay for IFN-γ-producing cells in patient RCC-A-03. The number of tumor-specific IFN-γproducing cells was undetectable when pre-vaccination peripheral blood lymphocytes were incubated with keyhole limpet... 42
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