Dendritic cells and dietary antigens [Elektronische Ressource] / vorgelegt von Maryna Nikulina

Nikulina

Sujets

• Gesundheit

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2006
• Nombre de lectures: 28
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Dendritic cells and dietary antigens



Inaugural-Dissertation
zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf



vorgelegt von
Maryna Nikulina
aus Temirtau


November 2006
Aus dem Deutsches Diabetes-Zentrum
an der Heinrich-Heine Universität Düsseldorf











Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf

Referent: Prof. Dr. H. Kolb
Koreferent: Prof. Dr. D. Riesner e.m.
Koreferent: Prof. Dr. D. Willbold

Tag der mündlichen Prüfung: 21.12.2006




Contents i
Contents

Contents ……………………………………………………………………………. i

Abbreviations……………………………………………………………………… iv

1 Introduction……………………………………………………………………… 1
1.1 Gut immune system……………………………………………………...1
1.2 Dendritic cells……………………………………………………………4
1.3 Oral tolerance………………………………………………………….. 12
1.4 Food antigens………………………………………………………….. 16
1.4.1 Wheat gluten……………………………………………………..16
1.4.2 Hsp 60……………………………………………………………19
1.5 Diabetes, food antigens and gut immune system……………………… 23
1.5.1 Type 1 diabetes mellitus (T1DM) ……………………………… 23
1.5.2 BB rats…………………………………………………………...24
1.5.3 Association between T1DM and celiac disease (CD) …………..25
1.5.4 Diet is a risk factor for T1DM………………………………….. 26
1.6 Aims of the current study……………………………………………… 28
2 Materials and methods………………………………………………………….29
2.1 Materials………………………………………………………………..29
2.1.1 Chemicals………………………………………………………. 29
2.1.1.1 Chemicals……………………………………………… 29
2.1.1.2 Inhibitors, agonists and antagonists…………………… 30
2.1.2 Materials for SDS-PAGE and Western blot……………………. 30
2.1.2.1 Antibodies……………………………………………… 30
2.1.2.2 Markers………………………………………………….31
2.1.2.3 Others…………………………………………………... 31
2.1.3 Antibodies for Fluorescence Activated Cell Sorting (FACS)
analysis…………………………………………………………. 31
2.1.3.1 Primary antibodies…………………………………….. 31
2.1.3.2 Secondary antibody……………………………………. 32
2.1.3.3. Blocking antibody……………………………………… 32
2.1.4 Materials for Enzyme-Linked Immunosorbent Assay (ELISA)... 32
2.1.4.1 ELISA Sets……………………………………………. 32
2.1.4.2 Others…………………………………………………. 34
2.1.5 Materials for Limulus Amebocyte Lysate (LAL) Assay………. 35
2.1.6 Materials for protein determination……………………………. 35
2.1.7 Medium, buffers and solutions………………………………… 35
2.1.7.1 Media and serum for cell culture……………………… 35
2.1.7.1.1 Media for dendritic cell culture……………. 35
2.1.7.1.2 Media for macrophage culture………………35 Contents ii
2.1.7.1.3 Investigated stimuli for dendritic
cell culture and macrophage culture……….. 36
2.1.7.2 Buffer for cell lysate preparation……………………… 37
2.1.7.3 Solutions, buffers and gels for SDS-gel
electrophoresis and electrotransfer…………………… 38
2.1.7.4 Solutions for immunological determination
of proteins by Western blot…………………………… 39
2.1.7.5 Solutions for FACS analysis………………………….. 39
2.1.7.6 Buffers and solutions for ELISA……………………… 39
2.1.8 Labware………………………………………………………… 41
2.1.9 Equipment……………………………………………………… 42
2.1.10 Animals………………………………………………………. 43
2.1.11 Diet…………………………………………………………… 43
2.2 Methods……………………………………………………………….. 44
2.2.1 Cell culture……………………………………………………… 44
2.2.1.1 Isolation and culture of dendritic cells (DCs)………….. 44
2.2.1.2 Culture of macrophages…………………………………45
2.2.2 Biochemical methods…………………………………………… 45
2.2.2.1 Solubilization of wheat gluten…………………………. 45
2.2.2.2 Isolation of gut tissue samples and preparation
of tissue homogenates………………………………….. 45
2.2.2.3 Preparation of cell lysates……………………………… 46
2.2.2.4 Protein determination………………………………….. 46
2.2.2.5 SDS-Polyacrylamide Gel Electophoresis
(SDS-PAGE) of proteins………………………………. 46
2.2.2.6 Electrophoretic transfer………………………………... 48
2.2.3 Immunological methods…………………………………………49
2.2.2.1 Immunological determination of proteins
by Western blot………………………………………… 49
2.2.3.2 FACS analysis…………………………………………. 49
2.2.3.3 ELISA…………………………………………………. 51
2.2.4 LAL assay………………………………………………………. 53
2.2.5 Statistics and graph computer programmes…………………….. 54
3 Results…………………………………………………………………………... 55
3.1 In vitro study of the effects of food components on innate immunity… 55
3.1.1 Wheat gluten……………………………………………………. 55
3.1.1.1 Wheat gluten stimulates maturation of BMDC………... 55
3.1.1.2 Wheat gluten-induced maturation of BMDC
is not due to bacterial contamination……………………56
3.1.1.3 Wheat gluten induces cytokine and
chemokine production in BMDC………………………..60
3.1.1.4 Wheat gluten-stimulated chemokine production
in BMDC is not secondary to IL-1β secretion………… 63 Contents iii
3.1.1.5 Wheat gluten signals in BMDC through p38,
extracellular signal-regulated kinases (ERK 1/2) and
NFκB, but not c-Jun NH -terminal kinase (JNK)……… 65 2
3.1.1.6 The active compound in wheat gluten preparation has
a peptidic nature………………………………………... 66
3.1.1.7 None of the investigated synthetic gliadin peptides
reproduce stimulatory activity of wheat gluten
α-chymotryptic digest on BMDC……………………… 67
3.1.1.8 PEP does not affect the stimulatory capacity of active wheat gluten peptide(s) on BMDC……………70
3.1.2 Hsp60…………………………………………………………… 71
3.1.2.1 Hsp60 induces maturation of BMDC………………….. 71
3.1.2.2 Hsp60 induces cytokine and chemokine
production in BMDC……………………………………73
3.1.2.3 HSP60-induced maturation of BMDC is partly due
to LPS………………………………………………….. 75
3.1.2.4 The involvement of TLR4 in Hsp60 signaling
in BMDC………………………………………………. 79
3.1.2.5 MAPK signalling in BMDC after stimulation
with Hsp60………………………………………………82
3.2 In vivo study of the effect of dietary wheat gluten on gut immunity…. 83
3.2.1 The level of IFN-γ in duodenum of BB rats fed the WG,
NTP-2000 or HC diets………………………………………….. 83
3.2.2 The level of IL-10 and of the ratio IFNγ/IL-10 in gut
duodenum of BB rats fed the WG, NTP-2000 or HC diets…….. 86
3.2.3 MCP-1 content in gut duodenum of BB rats fed the WG, 92