Dissertationen zum Thema „Immunogenetics“

Investigations have been made into the genetic control of immunity to the nematode Trichuris muris. Both background genes and genes within the mouse major histocompatibility complex (MHC), H-2, were shown to influence the expulsion of T. muris with the former having the stronger influence. At least two genes within the H-2 complex determined response phenotypes, the effects of "resistance" or "susceptibility" alleles at I-A being modulated by resistance or susceptibility alleles at aD end locus/loci. Differential responsiveness within slowly responding mouse strains suggested that parasite-dependent effects were also important. The primary antibody response to T. muris excretory/secretory (E/S) antigen, predominantly an IgG response, was also shown to be controlled by background and H-2-linked genes. In general, mouse strains less resistant to infection developed higher levels of IgG than- more resistant strains of mice. However strains of mice possessing the H-2q haplotype, irrespective of their genetic background, rapidly developed higher levels of IgG1 antibodies than strains of other haplotypes, H-2q haplotype mice tending to be more resistant to infection. Recognition of two high molecular weight (MW) E/S antigens by IgG as revealed by immunoprecipitation was also found to be almost exclusively H-2q restricted. This restriction may be partly quantitative but as such would operate in vivo due to the restriction on the ability to produce high levels of specific IgG. Both H-2q restricted phenomena may be part of, but not absolute requirements for, protective immunity. Parasite-induced effects on host immunity were also studied. Later larval and adult stages of T. muris were shown to be immunosuppressive, immunosuppression being long lasting and preventing the expulsion of subsequent infections. Vaccination with E/S antigen was shown to protect strains of mice which are slow to expel worms (poor-responder) or totally unable to expel worms (non-responder) from a primary infection with T. muris. However protection was slow to be expressed. Antigen recognition profiles of vaccinated strains of mice differed from their primary infection recognition profiles and included the recognition of the two high MW antigens shown to be H-2q restricted in a primary infection. Thus altering the mode or route of E/S antigen presentation may lead to shifts in responsiveness of H-2 genotypes to specific determinants and/or boost specific antibody levels sufficiently to reveal recognition of these antigens. Prior experience of a patent primary infection prevented vaccination protecting non-responder mice against subsequent infections. This inability was correlated with suppressed IgG1 antibody levels and failure to recognise three high MW antigens including the IL-2q restricted antigens. Using a panel of monoclonal antibodies raised against E/S antigen it was shown that E/S antigens, apparently including both immunogenic and immunosuppressive molecules, were localised to granules within the stichocyte cytoplasm of the adult T. muris stichosome.

La drépanocytose est l’hémoglobinopathie héréditaire la plus fréquente, causée par un polymorphisme unique d’un nucléotide (SNP) dans le gène de la beta-globine (HBB). Ce SNP détermine la synthèse de l’hémoglobine S, qui polymérise lorsqu’elle est soumise au stress, et ceci change la forme des hématies drépanocytaires en faucille. Les drépanocytes sont moins déformables, plus adhérents à l’endothélium, et plus susceptibles à l’hémolyse. Les complications cliniques de la drépanocytose peuvent être expliquées par l’interaction entre la vaso-occlusion, l’hémolyse et l’activation inflammatoire résultant de la présence des drépanocytes dans la circulation. Les patients drépanocytaires peuvent présenter de nombreuses complications, qui touchent tous les organes. La présentation clinique de cette maladie est très hétérogène, variant entre des patients qui ont très peu de symptômes à des patients qui décèdent de la maladie. Sachant que l’inflammation joue un rôle majeur dans la physiopathologie de la drépanocytose, des polymorphismes dans les gènes inflammatoires peuvent être évoqués pour expliquer cette hétérogénéité. La greffe de cellules souches hématopoïétiques est la seule thérapie curative disponible actuellement pour la drépanocytose, avec des bons résultats démontrés après la greffe d’un donneur apparenté HLA identique. Néanmoins, la plupart des patients n’a pas de donneur apparenté. Les patients drépanocytaires sont d’origine africaine, le groupe ethnique le moins représenté dans les registres de donneurs non apparentés de cellules souches. A ce jour, peu d’études, utilisant des registres locaux, ont été faites pour estimer la probabilité des patients drépanocytaires de trouver un donneur potentiel non apparenté dans les registres internationaux.Cette étude a eu pour objectif d’évaluer le rôle de quelques gènes inflammatoires liés aux Toll-like récepteurs (TLR) dans la survenue des infections bactériennes chez les patients drépanocytaires, vu que les infections sont une cause majeure de mortalité chez ces patients, et les TLR sont impliqués dans la reconnaissance de plusieurs types de bactéries. Les patients inclus avaient des échantillons d’ADN et des données cliniques disponibles. Les SNPs ont été génotypés par réaction en chaîne par polymérase en temps réel (RT-PCR). Quatre-cents trente patients, la plupart d’origine brésilienne ou africaine subsaharienne, ont été divisés en deux groupes : infectés (n=235, patients qui ont eu au moins un épisode d’infection bactérienne documentée) et non infectés (n=195, patients qui n’ont jamais présentés d’infections sévères). Le génotype T/A du SNP rs4696480 in TLR2 a été plus fréquent chez les patients non infectés (50% versus 67%, OR=0.50, 95% CI 0.34-0.75, p<0.001). En outre, le génotype T/T de ce SNP a été plus fréquent chez les patients infectés (15% versus 5%, OR=0.50, 95% CI 0.34-0.75, p<0.001). Des études précédentes ont démontré que les individus A/A avaient plus de sécrétion de marqueurs inflammatoires, lorsque l’allèle T était associé à moins de fréquence et de sévérité des maladies inflammatoires. Cette étude a également eu pour objectif d’estimer la probabilité de trouver un donneur potentiel non apparenté, antigène leucocytaire humain (HLA) allélique identique pour les loci HLA-A, HLA-B et HLA-DRB1. Dans cette étude, 185 patients ont été inclus, 116 suivis dans un centre brésilien et 69 greffés d’un donneur apparenté ou non apparenté dans des centres de greffe qui reportent leurs données à la Société Européenne de Greffe de Cellules Souches (EBMT). Les patients inclus avaient des données HLA testés en moyenne ou haute résolution. Les haplotypes HLA ont été estimés à travers un logiciel, HaploStats, et classifiés selon l’ethnicité. Ensuite, nous avons recherché des potentiels donneurs HLA alléliques identiques pour les loci HLA-A, HLA-B et HLA-DRB1 (6/6) dans des registres internationaux (WMDA)
Sickle cell disease (SCD) is the most common inherited hemoglobinopathy, caused by a single nucleotide polymorphism (SNP) in the beta-globin (HBB) gene. This SNP determines the synthesis of S haemoglobin (HbS), which polymerizes under stress conditions, sickling the red blood cell (RBC). Sickle RBC are less deformable, more adherent to the endothelium, and more susceptible to haemolysis. SCD complications are explained by the interaction between haemolysis, vaso-occlusion and inflammatory activation, determined by the RBC sickling. Patients with SCD may present several complications, affecting all organs. Clinical presentation is very heterogeneous, ranging from patients who have mild symptoms to patients who die from disease complications. Because inflammation plays a major role in SCD, polymorphisms in inflammatory genes are potential targets to explain this heterogeneity. Haematopoietic stem cell transplantation (HSCT) is the only curative therapy currently available for SCD, with good results shown after human leukocyte antigen (HLA) identical sibling HSCT. However, most patients will not have a matched sibling donor. Patients with SCD are mostly from African origin, the less represented ethnic group in stem cell donor registries. To date, few studies using local registries were performed to find the probability of having a potential unrelated donor in SCD settings. This study aimed to assess the role of inflammatory genes encoding Toll-like receptors (TLR) in the occurrence of bacterial infections in patients with SCD, because infection is a leading cause of mortality in SCD, and TLR recognize a wide range of bacteria. Patients included had DNA samples and clinical data available. SNPs were genotyped by real-time polymerase chain reaction (RT-PCR). Four hundred thirty patients, mostly from Brazilian and Sub-Saharan African origin, were divided in two groups: infected (n=235, patients who presented at least one episode of bacterial infection), and non-infected (n=195, patients who never presented bacterial infections). The T/A genotype of SNP rs4696480 in TLR2 was less frequent in infected patients (50% versus 67%, OR=0.50, 95% CI 0.34-0.75, p<0.001). In addition, the T/T genotype of this SNP was more frequent among infected patients (15% versus 5%, OR=0.50, 95% CI 0.34-0.75, p<0.001). Previous reports in other settings showed that A/A carriers had higher secretion of inflammatory markers, while T allele was associated with less occurrence and severity of inflammatory diseases. Hence, T/A genotype might express the ideal inflammatory response to defeat bacteria, while the weaker inflammatory response determined by the T/T genotype increases susceptibility to bacterial infections in SCD settings
A doença falciforme (DF) é a hemoglobinopatia hereditária mais frequente, causada por um polimorfismo de nucleotídeo único (SNP) no gene da betaglobina (HBB). A ocorrência desse SNP determina a síntese de hemoglobina S, que polimeriza sob condições de stress, alterando a conformação das hemácias, que adquirem forma de drepanócitos. Os drepanócitos são menos deformáveis, mais aderentes ao endotélio e mais suscetíveis à hemolise. As complicações clínicas da DF podem ser explicadas pela interação entre a vasoclusão, hemólise e ativação inflamatória resultantes da presença dos drepanócitos na circulação. Os pacientes com DF podem apresentar numerosas complicações, que afetam todos os órgãos. A apresentação clínica da DF é muito heterogênea, variando de pacientes pouco sintomáticos a pacientes que falecem por complicações da doença. Visto que a inflamação tem um papel importante na fisiopatologia da DF, polimorfismos em genes inflamatórios poderiam explicar essa heterogeneidade.O transplante de células tronco hematopoiéticas (TCPH) é a única terapia curativa disponível atualmente para a DF, com bons resultados demonstrados em TCPH de doador aparentado antígeno leucocitário humano (HLA) idêntico. Não obstante, a maioria dos pacientes não dispõe de doador aparentado HLA idêntico. A DF ocorre em pacientes normalmente de origem africana, o grupo étnico menos representado em registro de doadores de células tronco. Nos dias de hoje, poucos estudos, utilizando registros locais, avaliaram a probabilidade de encontrar potenciais doadores não aparentados para pacientes com DF. Este estudo teve por objetivo avaliar o papel de genes inflamaórios que codificam receptores Toll-like (TLR) na ocorrência de infecções bacterianas em pacientes com DF, visto que infecção é uma das principais causas de mortalidade em DF, e os TLR reconhecem diversos tipos de bactérias. Os pacientes incluídos no estudo tinham amostras de DNA e dados clínicos disponiveis. Os SNPs foram genotipados por reação em cadeia de polimerase em tempo real (RT-PCR). Quatrocentos e trinta pacientes, a maioria de orgem brasileira ou africana subsaariana, foram divididos em dois grupos, infectados (n=235, pacientes que apresentaram ao menos um episodio de infecção bacteriana), e não infectados (n=195, pacientes que nunca tiveram tais infecções). O genótipo T/A do SNP rs4696480 foi menos frequente em pacientes infectados (50% versus 67%, OR=0.50, 95% CI 0.34-0.75, p<0.001). Além disso, o genótipo T/T do mesmo SNP foi mais frequente em pacientes infectados (15% versus 5%, OR=0.50, 95% CI 0.34-0.75, p<0.001). Estudos prévios mostraram que indivíduos com genótipo A/A apresentavam mais secreção de marcadores inflamatórios, enquanto o alelo T foi associado a menor ocorrência e menor gravidade de doenças inflamatórias

[Truncated abstract] Natural killer (NK) cell alloreactivity can be exploited in haploidentical haematopoietic stem cell transplantation (HSCT) to improve graft survival, reduce graft versus host disease and decrease leukaemic relapse. NK cells lyse cells that have reduced expression of class I HLA molecules. In an allogeneic setting, donor NK cells may be activated by the absence of donor (self) class I HLA molecules on recipient cells; the absence of self-epitopes being detected by inhibitory KIR receptors on donor NK cells. The way in which genetic polymorphism of the receptors and ligands affects NK allorecognition of missing self, has not been fully elucidated. HLA-C molecules are divided into two groups, C1 and C2, with KIR2DL1 recognising cells expressing C2 and KIR2DL2 and KIR2DL3 recognising cells expressing C1. Donor NK cells expressing KIR2DL2 or KIR2DL3 can be alloreactive towards a recipient if they lack the C1 epitope and donor NK cells expressing KIR2DL1 can be alloreactive towards a recipient if they lack the C2 epitope. KIR3DL1 recognises the Bw4 epitope present on one-third of HLA-B alleles and certain HLA-A alleles. NK cells from donors expressing KIR3DL1 can be alloreactive towards recipients whose cells lack Bw4. Mismatches of KIR related HLA epitopes does not always results in NK alloreactivity. Therefore it is not possible to reliably predict NK alloreactivity based solely on the donor's HLA type and KIR repertoire and the recipient's HLA type. ... All Bw4-positive HLA-B alleles, with the exception of HLA-B*1301 and B*1302, protected targets from lysis. HLA-A*2402 and HLA-A*3201 unequivocally protected target cells from lysis whereas HLA-A*2501 and HLA-A*2301 provided only weak protection from lysis. KIR3DL1-dependent alloreactive NK clones were identified in donors whose only Bw4 positive allele was HLA-A*2402 but not in donors whose only Bw4 positive HLA allele was HLA-B*1301 or B*1302. Finally this thesis demonstrated that an activating KIR can control NK cell alloreactivity. Donors who are C2 negative and KIR2DS1 positive had NK cells that expressed the activating receptor KIR2DS1 and were capable of lysing cells expressing the C2 epitope. More so, KIR2DS1 dependent NK clones were shown to override inhibitory signals generated by NKG2A interacting with its ligand, HLA-E. The identification of these NK clones has important implications for haploidentical HSCT in that recipient expressing all three NK epitopes, C1, C2 and Bw4 were previously thought to be resistant to alloreactive NK cells controlled by inhibitory receptors. Such patients may be amenable to haploidentical HSCT from C2 negative, KIR2DS1 positive donors. These results will improve the ability to predict NK cell alloreactivity based on a donor's HLA type and KIR repertoire and the recipient?s HLA type.

Genetic susceptibility to type 1 diabetes is associated with the major histocompatibility complex (MHC) on human chromosome 6. To analyse the contribution of the class I region of the MHC to disease susceptibility, polymorphism of the class Ib molecule HLA-E and several genetic markers within the region was investigated in 378 British Caucasoid patients with type 1 diabetes as defined by the National Diabetes Group, 100 multiplex families from the BDA Warren Repository and 216 unselected normal British Caucasoid controls. PCR-Sequence-Specific Oligonucleotide Probing (PCR-SSOP) was used to analyse polymorphism of the HLA-E gene, Restriction Fragment Length Polymorphism analysis was used to investigate the four genomic probes P3A, P3B, 28L and 66R and finally microsatellite analysis to examine the two markers P 1 and D6S306. The association of the class I region with type 1 diabetes was also investigated with respect to gender and age at onset of type 1 diabetes. Linkage with known MHC class II susceptibility alleles was also analysed and the transmission disequilibrium test was used to analyse the BDA multiplex families. Significant increases in the frequency of the 1.5; 1.5 kilo base (kb) (Pc = 0.0004) and 1.8; 1.5 kb (Pc= 0.008) P3B genotypes were found in the patients compared to the controls. The P3B genotype 1.8; 1.8 was found to be decreased in the patients compared to the controls 26.6% vs 51.5% (Pc = 0.000002). This decrease in the 1.8; 1.8 P3B genotype was particularly apparent in those patients diagnosed before the age of 20 years. There were also differences in the frequency of the P3B genotype between males and female patients. The 1.5 P3B allele was increased and the 1.8 kb allele decreased in patients ( 46.1% and 53.9%) respectively compared to normal controls (27.8% and 72.1 %) respectively (Pc = <0.000001). The 1.8 P3B allele was increased in the <10 years compared to the 10- 20 years age group 60.2% vs 49.1% (Pc = 0.06). Further, the 1.8kb allele was increased in female patients with an age at onset of < 10 years compared to those in the 10-20 years group 63.5% vs 45.8% (Pc = 0.04). Analysis of the BDA multiplex families using the transmission disequilibirum test (TDT) showed that the 1.5kb allele was significantly transmitted from parents to affected offspring (Pc = 0.000002), suggesting that the 1.5 allele is in linkage with a susceptibility locus for type 1 diabetes. There was a highly significant difference in the observed transmission of the 1.5kb allele in the > 10 years group compared to the observed transmission of the 1.8kb allele in the > 10 years group (Pc = 0.000002). Increased frequencies of the HLA-E genotypes 0101;0101 (Pc = 0.0007), 0101;0103 (Pc = 0.025) were found in patients compared to controls. The HLA-E genotypes 0102;0104 (Pc= 0.001) and 0103;0104 (Pc= 0.004) were decreased in patients compared to controls. The 0101;0 101 genotype was increased in patients with an age at onset of 10-20 years compared to patients in the < 10 years group 4 7.0% vs 21 .1% (Pc = 0.001). The HLA-E 0101 allele was increased in patients compared to controls 54.1% vs 28.7% (Pc = 0.000003). In contrast, the alleles 0102 and 0104 were decreased in patients compared to controls (Pc = 0.000003, p = 0.0000 1) respectively. The 0 I 03 allele was increased in patients with an age at onset of < 10 years compared to the 10-20 years group 34.1% vs 18.9% (Pc = 0.02) in contrast, the 0101 allele was increased in the 10-20 years compared to the <10 years 62.9% vs 47.6% (Pc = 0.07). The frequency of the 0103 allele was increased in female patients in the < 10 years compared to the 10-20 years 37.5% vs 16.2% (Pc = 0.04). Haplotype analysis showed the significant presence of the P3A-P3B-28L-HLA-E (4-1.5-3 .8-0101) haplotype in patients with type 1 diabetes, which suggested the possible association between the P3B 1.5kb allele and the HLA-E 0101 allele. This was also supported by the presence of the 3 loci haplotype P3B-28L-HLA-E (1.5-3 .8-0101) that was found significantly present in patients. Combined genotype analysis of the P3B and HLA-E showed that the 0101-0103-1.5 genotype was significantly increased in patients compared to controls (Pc = 0.08). Analysis of the class lI 'diabetes-associated' alleles showed there was no significant linkage/association with either the P3B or HLA-E locus, suggesting that the class I association with type 1 diabetes may be independent of the class II. In conclusion a strong association with type 1 diabetes has been identified within the class I region, localised to the central area of the region. The association is related to gender and age at onset of type 1 diabetes, particularly the 10-20 years age at onset group. Identified HLA-E as a novel susceptibility gene, which is the first report of an association between a class Ib gene and an autoirnmune disease.

Genetic variation is crucial for the long-term survival of the species as it provides the potential for adaptive responses to environmental changes such as emerging diseases. The Major Histocompatibility Complex (MHC) is a gene family that plays a central role in the vertebrate’s immune system by triggering the adaptive immune response after exposure to pathogens. MHC genes have become highly suitable molecular markers of adaptive significance. They synthesize two primary cell surface molecules namely MHC class I and class II that recognize short fragments of proteins derived respectively from intracellular (e.g. viruses) and extracellular (e.g. bacteria, protozoa, arthropods) origins and present them to immune cells. High levels of MHC polymorphism frequently observed in natural populations are interpreted as an adaptation to detect and present a wide array of rapidly evolving pathogens. This variation appears to be largely maintained by positive selection driven mainly by pathogenic selective pressures. For my doctoral research I focused on MHC I and II variation in free-ranging cheetahs (Acinonyx jubatus) and leopards (Panthera pardus) on Namibian farmlands. Both felid species are sympatric thus subject to similar pathogenic pressures but differ in their evolutionary and demographic histories. The main aims were to investigate 1) the extent and patterns of MHC variation at the population level in both felids, 2) the association between levels of MHC variation and disease resistance in free-ranging cheetahs, and 3) the role of selection at different time scales in shaping MHC variation in both felids. Cheetahs and leopards represent the largest free-ranging carnivores in Namibia. They concentrate in unprotected areas on privately owned farmlands where domestic and other wild animals also occur and the risk of pathogen transmission is increased. Thus, knowledge on adaptive genetic variation involved in disease resistance may be pertinent to both felid species’ conservation. The cheetah has been used as a classic example in conservation genetics textbooks due to overall low levels of genetic variation. Reduced variation at MHC genes has been associated with high susceptibility to infectious diseases in cheetahs. However, increased disease susceptibility has only been observed in captive cheetahs whereas recent studies in free-ranging Namibian cheetahs revealed a good health status. This raised the question whether the diversity at MHC I and II genes in free-ranging cheetahs is higher than previously reported. In this study, a total of 10 MHC I alleles and four MHC II alleles were observed in 149 individuals throughout Namibia. All alleles but one likely belong to functional MHC genes as their expression was confirmed. The observed alleles belong to four MHC I and three MHC II genes in the species as revealed by phylogenetic analyses. Signatures of historical positive selection acting on specific sites that interact directly with pathogen-derived proteins were detected in both MHC classes. Furthermore, a high genetic differentiation at MHC I was observed between Namibian cheetahs from east-central and north-central regions known to differ substantially in exposure to feline-specific viral pathogens. This suggests that the patterns of MHC I variation in the current population mirrors different pathogenic selective pressure imposed by viruses. Cheetahs showed low levels of MHC diversity compared with other mammalian species including felids, but this does not seem to influence the current immunocompetence of free-ranging cheetahs in Namibia and contradicts the previous conclusion that the cheetah is a paradigm species of disease susceptibility. However, it cannot be ruled out that the low MHC variation might limit a prosperous immunocompetence in the case of an emerging disease scenario because none of the remaining alleles might be able to recognize a novel pathogen. In contrast to cheetahs, leopards occur in most parts of Africa being perhaps the most abundant big cat in the continent. Leopards seem to have escaped from large-scale declines due to epizootics in the past in contrast to some free-ranging large carnivore populations in Africa that have been afflicted by epizootics. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, I characterized genetic variation at MHC I and MHC II genes in free-ranging leopards from Namibia. A total of six MHC I and six MHC II sequences were detected in 25 individuals from the east-central region. The maximum number of sequences observed per individual suggests that they likely correspond to at least three MHC I and three MHC II genes. Hallmarks of MHC evolution were confirmed such as historical positive selection, recombination and trans-species polymorphism. The low MHC variation detected in Namibian leopards is not conclusive and further research is required to assess the extent of MHC variation in different areas of its geographic range. Results from this thesis will contribute to better understanding the evolutionary significance of MHC and conservation implications in free-ranging felids. Translocation of wildlife is an increasingly used management tool for conservation purposes that should be conducted carefully as it may affect the ability of the translocated animals to cope with different pathogenic selective pressures.
Genetische Variabilität ist entscheidend für das langfristige Überleben von Arten, denn es ermöglicht dem Organismus sich Umweltveränderungen, wie z.B. neu aufkommende Krankheiten, schneller anzupassen. Der Haupthistocompatibilitätskomplex (MHC) ist eine Familie von Genen, der eine zentrale Rolle im Immunsystem von Wirbeltieren zukommt, da sie nach Pathogenkontakt das adaptive Immunsystem aktivieren. Zudem sind MHC Gene geeignete molekulare Marker um Anpassungsfähigkeiten aufzuzeigen. MHC Gene kodieren primär für Zelloberflächenmoleküle, die kurze Peptidfragmente erkennen und den Immunzellen präsentieren, die im Falle der Klasse I Gene intrazellulären (z.B. von Viren) oder im Falle der Klasse II Gene extrazellulären (z.B. von Bakterien, Protozoen, Arthropoden) Ursprungs sein können. In der Regel wird in natürlich vorkommenden Populationen ein hoher Grad an Polymorphismus im MHC beobachtet, was als Anpassung an das Erkennen und Präsentieren einer großen Anzahl sich schnell entwickelnder Pathogene interpretiert wird. Das Bestehen vieler MHC Varianten über große Zeiträume hinweg wird hauptsächlich durch positive Selektion bewirkt, der ein pathogengetriebener Selektionsdruck zugrunde liegt. In meiner Doktorarbeit habe ich mich mit der Variation von MHC I and MHC II in freilebenden Geparden (Acinonyx jubatus) und Leoparden (Panthera pardus) in Farmgebieten innerhalb Namibias beschäftigt. Beide Felidenarten leben sympatrisch und sind so demselben Pathogendruck ausgesetzt, sie unterscheiden sich allerdings in ihrem evolutionären und demographischen Hintergrund. Mein Hauptziel war es 1) das Ausmaß und Muster der MHC Variation auf Populationsebene beider Feliden zu untersuchen; 2) einen möglichen Zusammenhang zwischen dem Grad der MHC Variation und der Krankheitsresistenz in frei lebenden Geparden aufzudecken und 3) zu untersuchen, welche Rolle der Selektion auf die MHC Variabilität beider Arten in der Vergangenheit wie auch gegenwärtig zukommt. Geparden und Leoparden repräsentieren die größten frei lebenden Carnivoren Namibias. Beide Arten kommen hauptsächlich in Farmgebieten vor, die sich in Privatbesitz befinden, und können dort mit anderen Wild- aber auch Haustieren zusammentreffen und potentiell Krankheitserreger austauschen. Die Kenntnis über die adaptive genetische Variation, die für Krankheitsresistenzen mitverantwortlich ist, kann für den Schutz beider Felidenarten von Bedeutung sein. Geparden werden häufig in Lehrbüchern als klassische Beispiele für eine Tierart mit einer generell geringen genetischen Diversität verwendet. Neben neutralen Markern ist bei Geparden auch eine geringe Variabilität der MHC Gene beschrieben worden, die als Ursache einer hohen Anfälligkeit für infektiöse Krankheiten gesehen wird. Bisher wurde allerdings eine erhöhte Krankheitsanfälligkeit nur bei Geparden aus Gefangenschaft beschrieben, wohingegen neuste Studien an frei lebenden Geparden diesen einen guten Gesundheitsstatus attestierten. Dadurch stellt sich die Frage, ob die MHC I und II Diversität in frei lebenden Geparden nicht höher sein könnte als bisher angenommen. In dieser Arbeit konnten insgesamt 10 MHC I und vier MHC II Allele in 149 frei lebenden Geparden aus ganz Namibia nachgewiesen werden. Die Zugehörigkeit zu funktionellen MHC Genen wurde durch Expressionsanalysen bei allen Allelen, außer einem, bestätigt. Durch phylogenetische Analysen konnten die Allele vier MHC I und drei MHC II Genen zu geordnet werden. Das Wirken von positiver Selektion in der Vergangenheit konnte an spezifischen Aminosäuren des Proteins, die in direktem Kontakt zu den pathogenen Antigenen stehen, festgestellt werden. Dies traf für beide MHC Klassen zu. Des Weiteren konnte eine starke genetische Differenzierung des MHC I zwischen Geparden aus einer nord-zentralen und einer ost-zentralen Region festgestellt werden, von denen auch bekannt ist, dass sie unterschiedlichen, felidenspezifischen, viralen Pathogenen ausgesetzt sind. Das lässt vermuten, dass die unterschiedlichen Muster der MHC I Variation in der gegenwärtigen Population den unterschiedlichen pathogengetriebenen Selektionsdruck durch Viren in den beiden Regionen widerspiegelt. Verglichen mit anderen Säugetierarten, insbesondere andere Feliden, zeigen Geparden einen geringen Grad an MHC Diversität, doch das scheint die derzeitige Immunkompetenz frei lebender Geparden in Namibia nicht einzuschränken und widerspricht der bisherigen Meinung dass Geparden ein typisches Beispiel für eine krankheitsanfällige Tierart sind. Es kann allerdings nicht ausgeschlossen werden, dass bei neu auftauchenden Krankheiten die geringe MHC Variation eine erfolgreiche Immunkompetenz verhindert, da möglicherweise keines der gegenwärtigen Allele die Fähigkeit besitzt neue Pathogene zu erkennen. Im Gegensatz zu Geparden kommen Leoparden in allen Teilen Afrikas vor und sind wahrscheinlich die am weitverbreiteste Großkatze des afrikanischen Kontinents. Es scheint, dass Leoparden, im Gegensatz zu anderen afrikanischen Großkatzen, einer ausgedehnten Dezimierung durch Tierseuchen in der Vergangenheit, der einige Populationen afrikanischer Großkatzen ausgesetzt waren, entkommen sind. Bisher fehlten Information über die MHC Variabilität in afrikanischen Leoparden. In dieser Studie konnte ich die genetische Variation der MHC I und MHC II Gene frei lebender namibischer Leoparden charakterisieren. In 25 Tieren aus einer Population der ost-zentralen Region konnten sechs MHC I sowie sechs MHC II Sequenzen nachgewiesen werden. Aus der maximalen Anzahl Allele pro Tier kann auf drei MHC I und auf drei MHC II Gene geschlossen werden. Außerdem konnten die typischen Kennzeichen einer variationserhaltenden MHC Evolution betätigt werden, wie positive Selektion in der Vergangenheit, Rekombination und über Artgrenzen hinaus bestehender Polymorphismus. Der geringe Grad an MHC Variation in namibischen Leoparden ist jedoch noch nicht endgültig und weitere Untersuchungen in unterschiedlichen Regionen aus der gesamten geographischen Verbreitung des Leoparden sind notwendig um die MHC Variation der Leoparden in Gänze einschätzen zu können. Die Ergebnisse dieser Arbeit werden zu einem besseren Verständnis des evolutionären Stellenwerts des MHC und in Folge zu einem besseren Schutz von frei lebenden Feliden beitragen. Die Umsiedelung von Wildtieren ist ein zunehmend angewendetes Hilfsmittel im Natur- und Artenschutz, welches jedoch mit Sorgfalt eingesetzt werden sollte, da die umgesiedelten Tiere möglicherweise einem anderen pathogenen Selektionsdruck ausgesetzt sind, dem sie nichts entgegenzusetzen haben.

(a) The class III region of the human MHC is approximately 1.1 Mbp in size. Almost 50% has been cloned in two overlapping cosmid clusters by chromosome walking from the complement/ 210H gene cluster and from the TNF a locus. The complement/ 210H cosmid cluster consists of 45 independent recombinants, spaning 390 kb. The TNFα and TNF β cosmid cluster consists of 16 independent recombinants, spanning 150 kb. Each genomic insert has been characterised by restriction enzyme digestion and Southern blot analysis to produce a detailed restriction map of the cloned region. (b) Single copy DNA sequences isolated from the cosmid clones have been hybridised to Southern blots of genomic DNA digests separated by pulsed-field gel electrophoresis (PFGE). This analysis has established the orientation and precise position of the complement/ 210H gene cluster within the class III region relative to the class I and class II genes. Furthermore, probes from the TNF cosmid cluster have been used to show that the TNF genes lie ~250 kb centromeric to the HLA-B locus. The gene order from the centromere is HLA-DR-210HB-C2-TNF α-TNF β-HLA-B, with HLA-OR separated from 210HB by 300-350 kb, and C2 separated from HLA-B by 650 kb. (c) Genomic probes have been used in conjunction with PFGE to define the positions of RTF islands within the cloned portion of the class III region. Single copy sequences associated with these islands have been hybridised to Southern blots of DNA from a variety of animal species to look for cross-hybridisation indicative of phylogenetic conservation of coding sequences. These probes have been used to screen Northern blots and define island related transcripts. The products of 15 genes have been mapped within the cloned region. Of these, two have been identified as duplicated members of the HSP 70 family. The remaining loci appear to be single copy sequences. One (Gll) has been cloned and characterised.

This thesis comprehensively applies genome-wide association study, Mendelian randomization analyses, and cytokine proteomics to investigate the genetic basis and immunopathogenic mechanisms of acute anterior uveitis (AAU). Multiple susceptibility loci, environmental risk factors, and potential biomarkers are identified, and a polygenic risk score for AAU developed. These findings provide novel insights into the immunogenetics in AAU, and contribute to clinical translational studies.

There are ~27 recognised extant species of crocodylians (Crocodylia), which inhabit a large range of tropical, fresh- or saltwater ecosystems around the world. Crocodylians appear to have an effective immune system that allows them to cope with a variety of pathogenic challenges. Crocodylians have been shown to live with a range of opportunistic infections, and yet wild animals seldom display disease symptoms. Also, reptile immune systems are less well studied than their mammalian and avian counterparts, and yet reptiles hold a unique evolutionary position as cold-blooded amniotes. Studying the evolutionary rates and diversity of immune system components in crocodylians could contribute to our overall understanding of the development of the immune system. Genetic and peptidome studies of the crocodylian immune system have improved our understanding of the organisation, evolution and diversity of some components of the adaptive immune system, however, there is much yet undiscovered when it comes to components that modulate the functions of the adaptive and innate immune systems in crocodylians. The availability of genomic resources would be beneficial to further investigations into disease susceptibility and resistance in industry and endangered wild populations. At present, only four crocodylian draft genomes have been sequenced and are publicly available. The projects undertaken in this thesis were initiated due to the limited genomic and immunogenetic resources available for crocodylian species. This research was to provide foundations in a key immune modulatory system in crocodylians, as well as to investigate applications of crocodylian genomic data in answering current evolutionary and immunogenetic questions.

Pharmacogenetics (PGx) aims at the definition of predictive and prognostic genetic biomarkers that can help clinicians in treatment tailoring. In this thesis, we explored two emerging topics in PGx: SNPs affecting the activity and maturation of a class of non coding RNAs, microRNAs (miRNAs), and immunogenetics. Genetic analyses were performed by a medium throughput technology, Veracode technology (Illumina). The clinical model of this study is represented by the locally advanced rectal cancer (LARC). Specifically, the main two aims of this study were: 1. the identification of predictive biomarkers of pathological response to neoadjuvant treatment in LARC patients, defined in terms of tumour regression grade (TRG); 2. the identification of biomarkers of disease free survival (DFS) and overall survival (OS) in LARC patients. To find an answer to these questions, we analyzed two panels of SNPs, selected according to bioinformatic analysis and literature data, on a group of 280 LARC patients homogeneously treated with fluoropyrimidines-based chemoradiotherapy in neo-adjuvant setting. In the first part of this project, we analyzed a panel of 144 SNPs potentially involved in miRNA maturation and activity. With a quite complex statistical strategy, we identified 5 new predictive biomarkers of response to neoadjuvant treatment. Specifically, DROSHA-rs10719 and SMAD3-rs17228212 were unfavourable predictive biomarkers (p=0.0274, p=0.0049), while SMAD3-rs744910, SMAD3-rs745103, and TRBP-rs6088619 showed an opposite effect (p=0.0153, p=0.0471, p=0.0125). Moreover, in patients with complete pathological response (TRG1), SMAD3-rs745103 was significantly associated with DFS (p=0.011). This study underlines the potential key role of SMAD3, factor involved not only in miRNA maturation but also in inflammation and in particular in TGFβ pathway, that is crucial in cancer progression and treatment response. Bearing in mind this interesting results and the huge amount of literature data addressing the high potentiality of immunogenetics in oncology, we analyzed a panel of 192 SNPs in genes involved in immune response in the same group of 280 LARC patients. We investigated another clinical end-point, the 2-year disease-free survival (2yDFS), because it is a strong prognostic biomarker of OS. Firstly, we identified 4 SNPs significantly associated with the 2yDFS. Two of them are located in IL17F (rs641701: OR=5.84, 95% CI=1.52-22.45, p=0.010; rs9463772: OR=3.56, 95% CI=1.22-10.35, p=0.020) and the other ones in STAT3 (rs8069645: OR=0.36, 95% CI=0.13-0.99, p=0.048; rs9867701: OR=3.00, 95% CI=1.09-8.30, p=0.034). Secondly, we studied the potential association of these 4 SNPs with the 10 years overall survival (OS). Interestingly, 3 SNPs remained significant and two of them are located in IL17F gene (IL17F-rs641701: OR=3.23, 95% CI=1.50-6.95, p=0.003; IL17F-rs9463772 OR=2.89, 95% CI=1.49-5.61, p=0.002, and STAT3-rs8069645 OR=0.50, 95% CI=0.25-0.98, p=0.044). We tested these associations in a validation group of 63 LARC patients who underwent radical surgery and adjuvant treatment based on fluoropyrimidines. Surprisingly, IL17F-rs9463772 is still significantly associated with OS (p=0.045), thus we can conclude that this is really a strong prognostic biomarker. To conclude, we performed 2 different PGx projects that led us to identify different predictive and prognostic biomarkers in LARC patients. These data, if confirmed in larger studies, will help clinicians to personalize patients treatment and management.

Type 1 diabetes is caused by immune destruction of pancreatic β cells. There is increasing evidence that genes outside the MHC region contribute to the pathogenesis of type 1 diabetes. Cytokines due to their role in immune regulation seem to play a crucial role in the pathogenesis of the disease. Three hundred and eight patients with type 1 diabetes and 150 normal controls were genotyped for polymorphism in the genes for IFN-γ, IL-4, IL-6, and TGF-β1. All assays employed in this study were PCR based. The IFN-γ CA repeats was an octa-allelic repeat and the 3 / 3 genotype showed a significant association with type 1 diabetes (p=0.0001). The IL-4 C (-590) T polymorphism did not show a significant association with type 1 diabetes. The GG genotype of G (-174) C of the IL-6 gene polymorphism showed a strong association with the susceptibility towards type 1 diabetes (p= 0.002). The TC genotype of the TGF-β1 T (+869) C polymorphism also showed a significant association with type 1 diabetes (p= 0.003). The association of the 3 I 3 genotype of the IFNG CA repeats and no association of IL-4 C (-590) T polymorphism may support the idea of dominance of the TH1 cytokine profile and type 1 diabetes suggesting a cell mediated disease. The IL-6 G (-174) C result attests an existing hypothesis of the important role of IL-6 in the onset of type 1 diabetes and its development. Immunosuppression of the TGF-β1 may have been initiated after deviation of the TH1/TH2 cytokine milieu. The GC of the IL-6 G (-174) C and the TC of the TGF-β1 T (+869) C showed strong association with diabetic nephropathy. Haplotype studies showed that cytokine function might be as a result of a cytokine network rather than individual cytokines. Further, the genetic susceptibility may be influenced not only by genetic composition but by the gender of patients as well as age at onset of type 1 diabetes. In conclusion these results suggest a contribution of the IFNG CA repeats, the TGF-β1 T (+869) C, and the IL-6 G (-174) C to the genetic susceptibility of type l diabetes and may have future therapeutical values.

Most of our understanding of immunity has been gained through studies of humans or laboratory rodents. However, such studies do not allow the immune system to be studied in the ecological context in which it has evolved and, as such, they provide a poor model for studying the variation in infectious disease resistance and immune function observed in natural settings. Studies of natural populations have provided fresh insights into the evolution and phenotypic consequences of immunogenetic variation, but have thus far concentrated almost exclusively on genes of the major histocompatibility complex (MHC). As these genes form only a fraction of the vertebrate immune repertoire, there is a need to broaden research in natural populations to include non-MHC genes, in order to gain a more comprehensive understanding of natural selection and immunity. In this thesis, the genetic diversity of a range of non-MHC immune genes was examined in a natural population of field voles (Microtus agrestis L.) in Kielder Forest, UK, which are subject to infection by a range of pathogens. Cytokine genes were the primary focus of this study as they play a central role in regulating the immune response but have rarely been studied in wild species. I examined the hypothesis that, as cytokines are crucial to immunity, variation within these genes may be under selection within populations and between species, and may mediate phenotypic differences between individuals in parasite resistance and immune function. Coding regions from nine cytokine and three other non-MHC vole genes were sequenced, yielding 6.6 Kb of sequence data and 26 SNPs (1 per 255 bp). Three cytokine genes (Il1b, Il2, and Tnf) exhibited patterns of polymorphism consistent with balancing selection maintaining genetic diversity, including an excess of intermediate frequency mutations and more even allele frequencies than one would expect under neutrality. Polymorphism within Il1b and Il2 was also consistently associated with variation in parasite resistance, providing evidence that pathogens are the selective force driving the maintenance of genetic diversity at these loci. In addition, Il1b and Il2 exhibited repeated associations with variation in host immune phenotype, while the Il12b gene was associated both with variation in pathogen resistance and with altered expression levels of Il1b and Il2. Variation in immune function, mediated through the cytokine network, is therefore likely to contribute to parasite resistance in the field vole. This work is the first to show that variation in cytokine genes of a natural population can be maintained by selection, and that this variation can lead to phenotypic variation in parasite resistance and immune function. More broadly, this thesis demonstrates that wild rodents are an excellent model to help us bridge the gap in our understanding between the mechanistic insights gained through studies on laboratory rodents and the variation in infectious disease susceptibility and immune function observed in nature.

Immune effectors are the cells responsible for host response to cancer cells and germs. There is a stringent need to understand immune effectors biology to improve infectious disease management, particularly for patients affected by deep grade immunosuppression. During my research, I chose to investigate the role of Glucose-6-Phosphate Dehydrogenase (G6PD) on immune effectors biology, to evaluate its relevance for response to infectious agents. G6PD is a key enzyme of pentose-phosphate pathway, producing reducing power in the form of NADPH, that is essential for oxidative stress protection machinery. The typical manifestations of enzyme deficiency are acute hemolytic anemia (AHA) following exposure to drugs or food and neonatal jaundice. The enzyme is expressed in all cells, but typical manifestations are related to enzyme deficiency in red cells. G6PD deficiency impact on white cells is less studied; some authors postulated that enzyme deficiency confers susceptibility to infections, but evidences are controversial. I tried to study the role of this enzyme defect in patients with hematologic malignancies, a population characterized by deep grade immunosuppression. Bacterial infections are the leading cause of morbidity and mortality in hematologic patients with neutropenia. Fluoroquinolone prophylaxis was proved to be the only effective strategy to reduce febrile neutropenia, but the safety of this class of agents in patients with G6PD deficiency is questionable because of a claimed association with AHA. I retrospectively analyzed 242 patients treated with 628 intensive chemotherapy courses; 59 patients presented G6PD deficiency. All patients underwent fluoroquinolone prophylaxis. The primary endpoint was incidence of AHA. Secondary endpoints were incidence of febrile neutropenia, microbiologically and clinically documented infections and incidence of Gram Positive or Gram Negative infections. No episode of AHA was observed in the entire cohort. Incidence of microbiologically and clinically documented infection was similar, but incidence of invasive fungal disease (IFD; p<0.0001, HR 11.4, 95%CI 3.5-37.05) and Candida sepsis (p=0.008, HR 37, 95% CI 2.01-680.9) was higher in patients with enzyme deficiency. Interestingly, I observed a reduced incidence of febrile neutropenia in patients with G6PD deficiency (p=0.01, HR 0.46, 95%CI 0.25-0.8). These data suggest that fluoroquinolone prophylaxis in patients with G6PD deficiency, treated with intensive chemotherapy, is feasible and safe. Also, data about IFD incidence and febrile neutropenia suggest that G6PD may be important in susceptibility to opportunistic pathogens and host response in neutropenic patients. Next, I evaluated G6PD deficiency role in susceptibility to IFD in patients affected by acute myeloid leukemia (AML) undergoing intensive chemotherapy, that are the patients at higher risk of fungal infections. I investigate this point in a cohort of 108 patients; 28 harbored G6PD deficiency, whereas 80 were normal. The incidence of IFD was significantly higher in patients with G6PD deficiency compared to normal patients (35.7% vs 5%, p=0.0002, OR=10, 95%CI=2.96-37.5). Higher risk of mold infections (17.9% vs 5%, p=0.048, OR=4.1, 95%CI=1.0-16.6) and Candida sepsis (17.9% vs 0%, p=0.0009, OR=37.68%, 95%CI=2.0-707.1) was observed in patients with G6PD deficiency. These data suggest that the evaluation of G6PD activity may help to identify AML patients at higher risk of IFD, allowing to design more intensive surveillance and therapeutic strategies. The identification of G6PD deficiency as a risk factor for IFD in patients with AML results in an urgent need for strategies to properly manage this kind of patients at high risk of invasive mycoses. Next, I proposed an algorithm for correct identification, prophylaxis and treatment of IFD in patients with G6PD deficiency undergoing intensive chemotherapy for AML.

This study describes the development and validation of mutagenized cloned DNA constructs, which correspond to the polymorphic regions of the class II region of the HLA complex. The constructs were used to verify the allelic specificity of primers and probes in polymerase chain reaction (PCR)-based HLA typing assays such as Sequence Specific Primers (SSP) and Sequence Specific Oligonucleotide Probes (SSOP). The constructs consisted of the entire polymorphic region of exon 2 of class II HLA allele sequences that included primer annealing sites or probe hybridization sites. An HLA allele sequence was inserted into a plasmid, cloned, then mutagenized to match a specific HLA allele, and finally, the correct clone was verified by bidirectional sequencing of the insert. Thus, the construct created a cloned reference DNA sample for any specific allele, and can be used to validate the accuracy of various molecular methodologies.