Host immune response in immunodeficient mice against infection with Cryptosporidium parvum [Elektronische Ressource] / Tesfaye Sisay Tessema

Sisay

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2008
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Host immune response in immunodeficient mice
against infection with Cryptosporidium parvum







Dissertation
zur Erlangung des Grades
“Doktor der Naturwissenschaften”



am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz


Tesfaye Sisay Tessema
geb. am 14.03.1974 in Dessie, Äthiopien

Mainz, 2008



































Tag der mündlichen Prüfung:10.12.2008
Table of contents

I. ntroduction 1

1. Pathobiology of Cryptosporidium parvum 1
1.1. Cryptosporidium species 1
1.1.1. Brief history 1
1.1.2. Taxonomy
1.1.3. Life cycle 2
1.2. Human and animal cryptosporidiosis 2
1.2.1. Epidemiology 2
1.2.1.1. Target population of cryptosporidiosis 3
1.2.1.2. The transmission stage: Oocyst 4
1.2.1.3. Transmof infection 4
1.2.1.4. Zoonosis 4
1.2.2. Clinical features and pathogenesis 5
1.2.3. Diagnosis 6
1.2.4. Control and prevention of cryptosporidiosis 6
1.2.4.1. Chemotherapy 6
1.2.4.2. Passive immunotherapy 7
1.2.4.3. Immune reconstitution
1.2.4.4. Prevention 8
1.2.5. Animal models of C. parvum infection 8
1.3. Host immune response to C. parvum 9
1.3.1. Innate immunity 9
1.3.1.1. Interferon- γ (IFN- γ) and natural killer (NK) cell in innate immunity 9
1.3.1.2. The action of phagocytes against C. parvum 10
1.3.1.3. Complement system 11
1.3.2. Adaptive immunity 12
1.3.2.1. Cell-medated immunity 12
1.3.2.2. Humoral immunity 18
1.3.3. Gut mucosal (local) immune system 19
1.3.3.1. Gut associated lymphoid tissues (GALT) 19
1.3.3.2. Intestinal epithelial cells (IECs) 20
i1.3.3.3. Intestinal intraepithelial lymphocytes (IELs) 22
1.3.4. Adoptive transfer of immunity and future prospects 24
+1.3.4.1. Immunological memory: memory CD4 T-cells 24
1.3.4.2. Adoptive transfer of immunity in C. parvum infection 26
1.4. Objectives of the thesis work 26

II. Materials and Methods 30

2. Materials 30
2.1. Parasites 30
2.2. Bacterial strain 30
2.3. Mouse strains
2.4. Nucleic acids 31
2.5. Equipment and Instruments 32
2.6. Consumables 33
2.7. Chemicals and Reagents 34
2.7.1. Chem 34
2.7.2. Antibodies 36
2.7.3. Enzymes 37
2.8. Media, Buffers and Solutions 37
2.8.1. Media for bacterial culture
2.8.2. Agarose gel electrophoresis buffer 37
2.8.3. Buffers and solutions used for cell isolation 38
2.8.4. Other buffers and solutions 39
3. Methods 40
3.1. Animal Experiments 40
3.1.1. Mouse infection protocol with C. parvum oocysts 40
3.1.2. Antibody treatments 41
3.1.3. Adoptive transfer of immune cells to naive mice 41
3.1.4. Animal sample collection 42
3.1.4.1. Tissue samples for RNA isolation 42
3.1.4.2. Tissue samples for genomic DNA isolation 42
3.1.4.3. Fecal sampling 42
3.1.4.4. Blood sam 43
ii3.2. Microbiological Methods 43
3.2.1. Culture of E. coli cels 43
3.2.2. Long-term storage and recovering of recombinant E .coli cells 43
3.2.3. Preparation of C. parvum oocysts for mouse infection 44
3.2.4. Determination of in vitro oocyst excystation rate (Test for viability) 44
3.3. Molecular Biological Methods 45
3.3.1. Preparation and analysis of nucleic acids 45
3.3.1.1. Total RNA isolation 45
3.3.1.2. Genomic DNA isolation from mouse tissues and cells 45
3.3.1.3. Plasmid DNA preparation 46
3.3.1.4. Extraction of DNA from agarose Gel 46
3.3.1.5. Determination of nucleic acid concentration 47
3.3.2. Primer design 47
3.3.3. Reverse transcription polymerase chain reaction (RT-PCR) 48
3.3.3.1. Reverse transcription 48
3.3.3.2. Polymerase chain reaction (PCR) 48
3.3.3.3. Electrophoresis of nucleic acids 49
3.3.4. Cloning of gene-specific PCR products 51
3.3.4.1. Generation of primer specific PCR products 51
3.3.4.2. Ligation with pJET1/blunt cloning vector 51
3.3.4.3. Preparation of competent E. coli cels 53
3.3.4.4. Transformation of competent E. coli cells 3
3.3.4.5. Analysis of transformed (recombinant) colonies 53
3.3.5. Designing external standard curves 55
3.3.6. Quantitative Real-time PCR 55
3.3.6.1. Assay design 55
3.3.6.2. Standard PCR protocol 56
3.3.6.3. Modified 57
3.3.6.4. Quantification analysis 58
3.3.6.5. Melting curve
3.4. Immunological Methods 59
3.4.1. Oocyst detection by immunofluorescence test (IFT) 59
3.4.2. Enzyme Linked Immunosorbent Assay (ELISA) 60
3.4.3. Separation of mouse lymphocytes for adoptive transfer 61
iii3.4.3.1. Isolation of mouse intestinal intraepithelial lymphocytes (IELs) 61
3.4.3.2. Separation of mouse spleen and MLN lymphocytes 65
3.4.3.3. Magnetic-activated cell sorting (MACS) of mouse lymphocytes 66
3.4.3.4. Flow Cytometry (FACS) 69
3.4.3.5. Homing of transferred lymphocytes to recipient mouse tissues 70
3.5. Software, data banks and web-based programs 70
3.6. Statistical Analysis 70

III. Results 71

4. Dynamics of Th1/Th2 cytokines in interferon-gamma and interleukin-12p40 KO
mice during primary and challenge Cryptosporidium parvum infections 71
4.1. Gene expression of Th1 and Th2 cytokines in the gut mucosa during
a patent primary C. parvum infection 72
4.2. Quantitative real-time RT-PCR assay design 74
4.2.1. Cloning of PCR products and analysis of transformants 74
4.2.2. Designing external standard curves 75
4.2.3. Quantification and melting curve analysis on LightCycler 77
4.3. Quantitative real-time PCR analysis of Th1/Th2 cytokine gene expression
changes during primary C. parvum infection 78
4.4. IFN- γ plays a role during early time of infection 80
4.5. TNF- α may have a regulatory role for the early IFN- γ response 80
4.6. Differential Th1 cytokine responses among mouse models 82
4.7. Expression of Th1 and Th2 cytokines during primary infection in mesenteric
lymph nodes (MLNs) and systemic lymphoid tissue (spleen) in GKO and
IL-12KO mice 82
4.8. Gene expression of Th1 and Th2 cytokines during challenge infection of
GKO and IL-12KO mice 84
4.8.1. The pattern of Th1/Th2 cytokines post challenge infection in gut mucosa 84
4.8.2. The pattern of Th1/Th2 cytokines post challenge infection in MLN
and splen 86
5. The role of interleukin-18 in resistance to C. parvum infection 87
5.1. Differential expression of IL-18 in the gut of C. parvum infected mice 88
5.2. In vivo neutralization of IL-18 increases the susceptibility of mice to infection 89
iv5.2.1. Systemic protein concentrations of IL-18 and IFN- γ 89
5.2.2. Influence of anti-IL-18 antibody on fecal oocyst shedding of mice 90
5.2.3. Influence of anti-IL-18 neutralizing antibody on gene expression
of cytokines 91
6. Adoptive transfer of immunity from C. parvum infected to naive mice 94
6.1. Characterization of isolated intestinal intraepithelial lymphocytes (IELs) 95
+6.1.1. Differential migration of CD4 T-cells to the gut mucosa between GKO
and IL-12KO mice at peak versus resolution of primary infection 96
+ +6.1.2. Population of CD4 and CD8 intestinal intraepithelial lymphocytes
transferred to naive recipients 97
6.2. Homing of adoptively transferred IELs to gut mucosal and systemic immune
tisues 99
6.3. Adoptive transfer of primed IELs provided protection to naive recipient mice
from C. parvum infection 101
6.3.1. Protection of naive GKO recipient mice from C. parvum infection was
conferred by day 15 p.i. donor IELs but not by day 8 p.i. donor IELs prepared
from GKO mice 101
6.3.2. Protection of naive IL-12KO recipient mice from C. parvum infection
was conferred by donor IELs as early as day 5 p.i. 103
+6.4. Adoptive transfer of primed CD4 T-cells provided protection to naive recipient
mice from C. parvum infection 105
6.4.1. Protection of naive GKO recipient mice from C. parvum infection was
+conferred by 8 and 15 days p.i. CD4 T-cells prepared from tissues of GKO
donor mice 105
6.4.2. Protection of naive IL-12KO recipient mice from C. parvum infection
+was conferred by 5 and 15 days p.i. CD4 T-cells prepared from tissues of
IL-12KO mice 107
+6.5. Naive CD4 T-cells and IELs do not provide protection to naive recipient mice 108
6.6. Adoptive transfer of pan T-cells provided equivallent level of protection with
+that of CD4 T-cells 109
6.6.1. Rationale for the experiment