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The immunomodulatory impact of interferon-alpha in combination with chemoradiation of pancreatic adenocarcinoma (CapRI) [Elektronische Ressource] / submitted by Emilia-Maria Patrut

57 pages
From the Surgical ClinicRuprecht-Karls-University of Heidelberg(Academic Director: Prof. Dr. med. Markus W. Büchler)IMMUNOMODULATORY IMPACT OF INTERFERON-ALPHA INCOMBINATION WITH CHEMORADIATION OF PANCREATICADENOCARCINOMA (CapRI)Inaugural ThesisFor the title of Doctor of medicineof theMedical FacultyRuprecht-Karls-University of Heidelbergsubmitted byEmilia-Maria PatrutfromCluj-Napoca2005Dean: Prof. Dr. med. Claus R. BartramReferee: Prof. Dr. med. Jan SchmidtCONTENTSPageABBREVIATION LIST 51. INTRODUCTION 71.1 Pancreatic cancer – epidemiology and prognosis 71.2 Adjuvant treatment of pancreatic adenocarcinoma 81.3 9Role of interferon-alpha (IFN-α) in the combined modality treatment ofpancreatic adenocarcinoma1.4 Antitumoral immune mechanisms 101.5 Mechanisms of apoptosis induction 121.6 Pathways of nuclear transcription factor NF-κB activation and its role in 15carcinogenesis1.7 Aims of the study 162. MATERIALS AND METHODS 172.1 Materials 172.1.1 Chemicals 172.1.2 Antibodies 182.1.3 Nucleotides 182.1.4 Pancreatic carcinoma cell lines 182.1.5 Blood preparations 182.1.6 Solutions and buffers 192.1.7 Culture medium 192.1.8 Other materials 192.2 Methods 202.2.1 Cell culture 202.2.2 Treatment of the pancreatic carcinoma cell lines 202.2.3 Preparation of lymphocytes 202.2.4 Flow cytometric analysis 212.2.5 MTT assay for cell proliferation 222.2.6 Determination of apoptosis, cell death and cell count 222.2.
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From the Surgical Clinic Ruprecht-Karls-University of Heidelberg (Academic Director: Prof. Dr. med. Markus W. Büchler)
IMMUNOMODULATORY IMPACT OF INTERFERON-ALPHA IN COMBINATION WITH CHEMORADIATION OF PANCREATIC ADENOCARCINOMA (CapRI)
Inaugural Thesis For the title of Doctor of medicine of the Medical Faculty Ruprecht-Karls-University of Heidelberg
submitted by Emilia-Maria Patrut
from Cluj-Napoca
2005
Dean: Prof. Dr. med. Claus R. Bartram
Referee: Prof. Dr. med. Jan Schmidt
CONTENTS
ABBREVIATION LIST
1. INTRODUCTION 1.1 Pancreatic cancer  epidemiology and prognosis 1.2 Adjuvant treatment of pancreatic adenocarcinoma 1.3 Role of interferon-alpha (IFN-α) in the combined modality treatment of pancreatic adenocarcinoma 1.4 Antitumoral immune mechanisms 1.5 Mechanisms of apoptosis induction 1.6 Pathways of nuclear transcription factor NF-κB activation and its role in carcinogenesis 1.7 Aims of the study
2. MATERIALS AND METHODS 2.1 Materials 2.1.1 Chemicals 2.1.2 Antibodies 2.1.3 Nucleotides 2.1.4 Pancreatic carcinoma cell lines 2.1.5 Blood preparations 2.1.6 Solutions and buffers 2.1.7 Culture medium 2.1.8 Other materials 2.2 Methods 2.2.1 Cell culture 2.2.2 Treatment of the pancreatic carcinoma cell lines 2.2.3 Preparation of lymphocytes 2.2.4 Flow cytometric analysis 2.2.5 MTT assay for cell proliferation 2.2.6 Determination of apoptosis, cell death and cell count 2.2.7 Cytotoxicity assays for untreated pancreatic carcinoma cells 2.2.8 Cytotoxicity assays for pretreated pancreatic carcinoma cells
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2.2.9 Blockage of apoptosis induction mechanisms 2.2.10 Granzyme B ELISPOT 2.2.11 Electrophoretic mobility shift assay for NF-κB binding activity 2.2.12 Statistical analysis
RESULTS 3.1 Comparison between chemoradiation and chemoradioimmunotherapy regarding the proliferation rate and apoptosis induction 3.2 The influence of IFN-αon lymphocytes 3.3 Analysis of the killing mechanism 3.4 Cytotoxic activity of IFN-αprestimulated lymphocytes against pretreated tumor cells 3.5 Cytotoxicity in the CapRI scheme versus chemoradiotherapy (CR) 3.6 The influence of IFN-α on the immunogenicity of pancreatic carcinoma cells 3.7 Cytotoxic activity of T cells against IFN-αtreated tumor cells 3.8 Effect of IFN-αon the NF-κB binding activity
DISCUSSION
SUMMARY
REFERENCES
CURRICULUM VITAE
ACKNOWLEDGEMENTS
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ABBREVIATION LIST 5-FU  5-Fluorouracil ADCC  antibody-dependent cellular cytotoxicity APC  antigen presenting cells BCA  Bicinchoninic acid BFA  Brefeldin A CAD  caspase activated deoxyribonuclease CapRI  adjuvant treatment of pancreatic adenocarcinoma with chemoradioimmunotherapy CD  clusters of differentiation CDDP  Cisplatin CMA  Concanamycin A CR  chemoradiation CTL cytotoxic T cells DC dendritic cells DISC  death inducing signalling complex EBV  Epstein-Barr virus EGF-R  endothelial growth factor receptor ELISA enzyme-linked immunosorbent assay ELISPOT  enzyme-linked immunospot EMSA  electrophoretic mobility shift assay ER  endoplasmic reticulum FADD  Fas associated death domain FasL  Fas ligand FITC  fluorescein isothiocyanate Gy  gray HLA  human leukocyte antigen IFN  interferon IFN-α- interferon-alpha IKK  IκB kinase IL  interleukin KIRs  killer inhibitory receptors LFA  lymphocyte function-associated antigen MACS  magnetic activated cell sorting MHC  major histocompatibility complex
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