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  Autoimmunity as a possible limiting selection pressure for the individual MHC IIB allele diversity in the three-spined stickleback Gasterosteus aculeatus?

Krause, A. (2011). Autoimmunity as a possible limiting selection pressure for the individual MHC IIB allele diversity in the three-spined stickleback Gasterosteus aculeatus? Diploma Thesis, Christian-Albrechts-Universität, Kiel.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-000F-D40F-F Version Permalink: http://hdl.handle.net/11858/00-001M-0000-000F-D410-9
Genre: Thesis

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Krause, Anja1, Author              
Milinski, Manfred2, Advisor              
Schulenburg, Hinrich, Referee
Affiliations:
1Department Ecophysiology, Max Planck Institute for Limnology, Max Planck Institute for Evolutionary Biology, Max Planck Society, ou_976547              
2Department Evolutionary Ecology, Max Planck Institute for Evolutionary Biology, Max Planck Society, ou_1445634              

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 Abstract: Genetic diversity is a prerequisite for evolution. The genes of the Major Histocompatibility Complex (MHC) show genetic variation. They are polygenic and contain highly polymorphic loci. MHC molecules are an important part of the adaptive immune system due to their ability to bind and present different antigens to the T-lymphocytes. But this high specificity also implies a risk: the higher the number of recognized antigens, the more likely the similarity of foreign and auto antigens. This can cause a loss of self tolerance mechanisms due to cross reactions during molecular mimicry. The MHC might be a key molecule for selection, adaptive processes and speciation. Gasterosteus aculeatus shows an intermediate number of MHC IIB alleles. On the one hand, too few different MHC IIB alleles demonstrate an insufficient diversity to detect a large number of parasite antigens. But on the other hand, there must be a limiting selection pressure, which causes this intermediate optimum of MHC IIB alleles. A possible disadvantage assumes that too many alleles might lead to self-immunoreactivity. Therefore, this thesis focuses on a possible correlation between certain immunological changes, which might be associated with autoimmune reactions, and the individual MHC IIB allele diversity of the three-spined stickleback. It is a pilot study about auto aggressive reactions as a possible limiting selection pressure on the evolution of the MHC IIB allele diversity. Thus, I used different methods to examine different aspects of this topic. First, the MHC class IIB allele configuration of parental fish of 14 different families was determined. The results indicate five families, whose offspring showed a MHC class IIB allele diversity of either two or four different alleles. The MHC genotyping of parents and offspring was done via the Reference Strand-mediated Conformation Analysis (RSCA). The next step was the isolation of several inflammatory genes. Different primer combinations were tested via standard PCR and Agarose gelelectrophoresis. After that, primer specificity was checked via direct sequencing and quantitative Real Time PCR. I was able to isolate IL 1 beta, MIF, IgM and MHC II in the genome of Gasterosteus aculeatus. After that, head kidney, spleen, liver, kidney, gut, gonads, heart and brain were dissected and blood was taken from the caudal vein. The total immunoglobulin level of the plasma was measured with the help of an Enzyme-linked immunosorbent assay (ELISA). RNA of head kidney, gut, kidney and spleen was isolated and reverse transcribed into cDNA. The expression levels of the established inflammatory genes MIF, IL 1 beta, IgM and MHC class II were measured, relative to those of the housekeeping genes L13 and Ubiquitin. This was done via the quantitative Real Time PCR, based on the non-specific reporter molecule SYBR Green I. The raw gene expression data was normalized with the software qBase Plus and a permutational multivariate analysis of variance was performed with the help of the software R 2.11.1. The results indicate the family of origin and sex as influencing factors. The influence of the dissection day is due to an artefact. Maybe different families contain different repertoires of immunological factors. This might be due to differences in their genetic background. In humans, it has been assumed that autoimmunity is correlated with female sex hormones. Therefore, it might be possible that female sex hormones of teleost fish are correlated with the occurrence of autoimmune diseases. It would be interesting to examine, whether and how female sex hormones can influence the adaptive immune system. Due to a low p-value (p = 0.074), it might be possible that the number of MHC IIB alleles also affects certain immunological parameters. Therefore, it might be interesting to breed fish with very high and low numbers of MHC IIB alleles and to expose them to many parasites and many different types of parasites. Maybe the costs, namely the occurrence of auto aggressive reactions, are lower than the benefits, namely a high resistance towards parasites. It might be possible that there is a trade-off between the resistance towards parasites and auto aggressive reactions The results of the expression assay indicate that IL 1 beta seems to be an important component of the adaptive immune response in Gasterosteus aculeatus. It shows a higher expression in the head kidney of individuals with four MHC IIB alleles. Apart from that, a positive correlation of IL 1 beta- and MHC II expression is observed in head kidney, spleen and kidney. It might also be helpful to detect more possible inflammatory genes for a better understanding of the complex network of associations between several cytokines of the adaptive immune system. Some studies about cytokines in teleost fish include stimulations of the adaptive immune system with LPS, FCA and ATH. They resulted in an increase of expression levels in several organs. Therefore, another interesting project might deal with the artificial induction of autoimmune reactions due to stimulation of the immune system of Gasterosteus aculeatus.

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Language(s): eng - English
 Dates: 2011-02
 Publication Status: Accepted / In Press
 Pages: 91 Bl.
 Publishing info: Kiel : Christian-Albrechts-Universität
 Table of Contents: Table of contents
1. Zusammenfassung ........................................................................................................... 5
2. Summary ........................................................................................................................... 7
3. Introduction ...................................................................................................................... 9
3.1 Evolution and the MHC polymorphism ..........................................................................9
3.2 Immune system and autoimmunity ............................................................................. 11
3.3 Cytokines – multifunctional regulators of adaptive immune responses ....................... 15
3.4 Immunoglobulins – the organisms arsenal against antigens ....................................... 16
3.5 Lymphatic organs – origin of organisms immunity ...................................................... 16
3.6 The Major Histocompatibility Complex (MHC) – initial element of the adaptive immune
response .......................................................................................................................... 17
3.7 The three-spined stickleback Gasterosteus aculeatus – an emerging model organism
of evolutionary genetics .................................................................................................... 18
3.8 Thesis outline ............................................................................................................. 19
4. Material and Methods ..................................................................................................... 20
4.1 Origin and MHC diversity of fish specimens used in the study .................................... 20
4.2 Organs, which were analyzed during the study ........................................................... 20
4.3 MHC class IIB - Genotyping ........................................................................................ 21
4.4 Examination of inflammatory genes by PCR, sequencing and sequence analysis ...... 23
4.4.1 Reverse Transcription PCR ................................................................................. 26
4.4.2 Standard PCR ...................................................................................................... 26
4.4.3 DNA Gel extraction .............................................................................................. 27
4.4.4 Direct Sequencing ............................................................................................... 27
4.5 Dissection of the examined three-spined sticklebacks ................................................ 28
4.6 Enzyme-linked immunosorbent assay (ELISA) ........................................................... 30
4.7 Quantitative Real Time PCR ....................................................................................... 31
4.7.1 RNA isolation ....................................................................................................... 31
4.7.2 RNA purity test ..................................................................................................... 31
4.7.3 Reverse Transcription PCR ................................................................................. 32
4.7.4 The quantitative Real Time PCR .......................................................................... 32
4.8 Statistical analysis ...................................................................................................... 34
4.8.1 PCR efficiency calculation by LinReg PCR .......................................................... 34
4.8.2 Normalization of gene expression data using qBase Plus .................................... 34
4.8.3 Descriptive statistics ............................................................................................ 35
4.8.4 Permutational multivariate analysis of variance (permutational MANOVA) ........... 36
5. Results ............................................................................................................................ 38
4
A. Previous requirements for the actual data analysis .................................................... 38
5.1 MHC class IIB-Genotyping.......................................................................................... 38
5.2 Examination of inflammatory genes by PCR and DNA sequencing ............................. 39
5.3 RNA purity test ........................................................................................................... 42
5.4 Quantitative Real Time PCR efficiency ....................................................................... 43
5.5 Stability of reference gene expression ........................................................................ 44
B. Main statistical analysis ................................................................................................ 47
5.6 Descriptive statistics ................................................................................................... 47
5.7 Permutational multivariate analysis of variance .......................................................... 48
5.7.1 Main analysis ....................................................................................................... 48
5.7.2 Permutational multivariate ANOVA according to the dissection day ..................... 50
5.7.3 Coefficients of the main analysis .......................................................................... 50
5.7.4 Gene expression analysis .................................................................................... 51
5.7.5 Permutational multivariate ANOVA for different organs ........................................ 54
5.7.6 Correlation between number of MHC IIB alleles and the expression of MHC II .... 55
5.7.7 Correlation between expression of Interleukin 1 beta and MHC II ........................ 55
6. Discussion ...................................................................................................................... 57
6.1 Methodological discussion .......................................................................................... 57
Examination of target genes – PCR and Sequencing ....................................................... 57
6.2 Interpretation of the results ......................................................................................... 59
6.2.1 Main statistical analysis – permutational multivariate ANOVA .............................. 59
6.2.2 Gene expression assay ....................................................................................... 61
6.3 Final conclusions ........................................................................................................ 64
6.4 Future prospects ......................................................................................................... 65
7. Acknowledgements ........................................................................................................ 68
8. References ...................................................................................................................... 69
9. Supplement ..................................................................................................................... 79
 Rev. Method: -
 Identifiers: eDoc: 529883
Other: Dipl/12268
 Degree: Diploma

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