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My dissertation hypothesis is that bovine trophoblast cells express cell-surface and secreted non-classical major histocompatibility complex class I (MHC-Ib) proteins which inhibit NK cells and other leukocytes by binding to inhibitory receptors (e.g., LILRB1, LILRB2, KIR2DL4, and/or CD94/NKG2A). Extremely polymorphic and ubiquitously expressed classical MHC class I (MHC-Ia) proteins, which present foreign antigenic peptides to CD8+ T lymphocytes, are involved in acceptance or rejection of tissue grafts. Non-classical MHC class I (MHC-Ib) glycoproteins, such as Human Leukocyte Antigen-G (HLA-G) and murine Qa-2, are important modulators of the maternal immune system during pregnancy. MHC-Ib proteins are: (a) oligomorphic or monomorphic, (b) expressed in specific tissues under specific condtions, and (c) produced as surface and/or soluble isoforms due to alternative splicing. Third trimester-bovine trophoblast cells express both MHC-Ia and MHC-Ib proteins. The MHC-Ib proteins expressed by trophoblast cells during the third trimester of pregnancy are encoded by four bovine leukocyte antigen (BoLA) loci: BoLA-NC1, BoLA-NC2, BoLA-NC3, and BoLA-NC4. Two MHC-Ia (N*01701 and N*01802) and three MHC-Ib (NC1*00501, NC3*00101 and NC4*00201) proteins showed cell-surface expression in transfection studies performed in murine P815 and human K562 cells. Two additional isoforms, NC1*00401 and NC2*00102, were not detected on the surface of these cells. Nevertheless, both class Ia proteins, N*01701 and N*01802, and five class Ib proteins, NC1*00401, NC1*00501, NC2*00102, NC3*00101, and NC4*00201, were detected in crude cell lysates on Western blots. Precipitation of proteins from culture supernatants showed that cell-surface MHC-Ia (N*01701 and N*01802) and MHC-Ib proteins (NC1*00501, NC3*00101, and NC4*00201) are shed from the surface of these cells into the media. The mechanism of shedding of these proteins is, however, not known. Monoclonal antibodies W6/32, IL-A88, H1A, H6A, H11A, H58A, and PT-85A recognized surface MHC-I isoforms with varying affinity. We were able to develop a sandwich enzyme-linked immunosorbent assay (ELISA) using either H1A or IL-A88 antibody as the capture antibody and the W6/32 antibody for detection. We produced monoclonal antibodies against cattle NC1*00501 and NC3*00101 proteins. One monoclonal antibody generated against BoLA-NC3*00101 was highly specific. Unfortunately, due to failure to clone the NC3*00101- hybridoma, we no longer have an infinite source of this monoclonal antibody for NC3*00101. We eluted peptides from NC3*00101-transfected MHC-null K562 cells and identified peptides using liquid chromatography-mass spectrum (LC-MS) analysis. Analysis of peptide binding data using the SAS Proc mixed statistical program, suggested that the peptide EVTNQLVVL is a potential peptide ligand, which can be used to make tetramers for enumeration of antigen-specific leukocytes.

The blue shark (Prionace glauca) is a highly migratory pelagic elasmobranch that inhabits ocean basins globally. As a result, this shark is exposed to intensive ocean exploitation by commercial target fisheries, by-catch and for recreational pursuits globally. This top predator is therefore at high risk of becoming overfished. Advances to current knowledge of genetic population structure and diversity of this species would provide vital information required to initiate co-operative management approaches. In this study, the major histocompatibility complex (MHC) class IIa and IIβ genes were successfully isolated and characterised from blue sharks. Phylogenetic trees of the class II genes showed three major clades; one of teleost fish, one of tetrapods and one of sharks. The MHC class IIβ gene exon 2 primers successfully amplified partial sequences in blue sharks from several global locations. Analysis of sequences using denaturing gradient gel electrophoresis (DGGE) suggested the assay resolved different sequences up to one basepair, making the assay potentially very useful with further development. The class II genes presented in this study show conflicting evidence for the presence of more than one class II locus. To explore inheritance patterns of MHC exon 2 diversity, a single blue shark litter (mother + 19 pups) was cloned and sequenced, revealing evidence to suggest the possibility of more than one locus for class IIβ. Statistical analysis of parasite loads and diversities from blue shark spiral valves revealed no definitive population structure, supporting global and North Atlantic mtDNA and microsatellites genetic analyses presented here. The size (fork length) of sharks was found to be potentially influential when modelled with individual microsatellite heterozygosity and fork length. International co-operation will be required to prevent this species becoming extinct from global marine ecosystems. Reductions in numbers could lead to reduced genetic diversity, decreased immunity and ultimately an 'unhealthy' population.

Being a dissertation presented in fulfilment of the requirements governing the degree of Masters of Science in the Faoulty of Medicine, University of the Witwatersrand / Recent investigations have shown enhanced or aberrant expression of major histocompatibility system (MHC) antigens on cells lines derived from human hepatocellular carcinoma (HCC) in vitro and HCC in vivo. ( Abbreviation abstract ) / AC2017

The pathogenesis of autism has proven difficult to characterize. However, in many recent studies, it is suggested that the onset of this disorder is the result of multiple etiological factors, which include genetic, immunologic, and viral elements. Possible immunological influences found in subpopulations of patients with autism include decreased lymphocyte responsiveness, reduced natural killer cell activity, abnormal response to rubella vaccine, abnormal immune response to brain tissue, and decreased plasma levels of the fourth component of complement(C4). These aberrations and others imply a possible autoimmune mechanism in some cases for the development of autism. C4 deficiencies have been found in subjects with established autoimmune disorders, such as systemic lupus erythematosus and chronic active hepatitis, in recent investigations. There is also evidence that the major histocompatibility genes play an intimate role in autoimmune processes. Therefore, in knowing that the C4 genes are closely linked to the major histocompatibility genes, this study determined and analyzed the human leukocyte antigen profile of autistic patients, their siblings, and parents. In this study, it was found that the C4B complement null allele occurred in autistic patients at nearly twice the frequency compared to normals. However, the C4A complement null allele frequency was not found to be significantly altered. Several extended haplotypes were represented within the patients studied. However, the extended haplotype B44- SC30-DR4 was the only one found at a significantly increased frequency. Further investigations are needed to better understand the significance of these findings.

The major histocompatibility complex (MHC) is a chromosomal region encoding molecules controlling adaptive immune response in vertebrates. In farm animals, many associations between MHC loci and productivity traits including disease susceptibility have been described. However, current knowledge about the structure and function of the MHC in domestic animals, especially sheep, is very limited. Characterization of the sheep MHC may potentially facilitate breeding for enhanced disease-resistant animals through use of marker assisted selection. The main aim of this project has been to provide insights into the organization of the genomic content of the central region of the sheep MHC. The work described herein has utilized subcloning of a sheep BAC genomic library in conjunction with DNA sequencing to generate a map of the central region of the sheep MHC covering ≈700 kbp. Within this map the relative order and identity of twenty five recognized loci were established. For some loci the intergenic distances were also determined. The final map is the most accurate map of this region reported to date and shows a high degree of similarity to the analogous region of the human MHC. This work has been published and a copy of the paper is included in Appendix 1. During the course of this work detailed genomic sequences were obtained for several sheep central region loci. Complete nucleotide sequences were generated for the complement factor B locus (CFB) and the TNFα locus and a comparative analysis of these sequences confirmed their homology with other vertebrate orthologues. Extensive partial sequences for complement components C2 and C4 were also obtained and reported to GenBank. / In addition, a previously identified short tandem repeat locus designated BfMs believed to be in the CFB locus was mapped to an intron within the adjacent SKI2VL locus. Single nucleotide polymorphisms (SNPs) were identified by analysing homologous sequences from a minimum of five individual sheep. In total 33 SNPs were discovered distributed over eleven distinct loci. Allele frequencies for SNPs from ten of these loci were determined and reported for a panel of 71 sheep comprising 58 unrelated sheep from the Rylington Merino flock plus a further 13 unrelated parental animals from a three generation half sibling sheep pedigree. The availability of an independently confirmed pedigree constructed from a three generation half sibling sheep family permitted the identification by deduction of central region MHC haplotypes based on a panel of SNPs derived from 10 loci. This is the first reporting of haplotypes covering this region of the sheep MHC. Analysis of SNP panel genotypes in the cohort of 71 unrelated sheep using the expectation maximization algorithm permitted the prediction of a group of approximately 20 haplotypes, which accounted for more than 90% of the expected haplotype distribution. Four of these predicted haplotypes were also present in the known haplotype cohort deduced from the sheep pedigree. Analysis of pairwise linkage disequilibrium between SNP loci in the cohort of 71 unrelated sheep showed a centre-most region displaying relatively high levels of linkage disequilibrium which was bounded by two regions displaying more variable linkage disequilibrium. / It is hypothesised that this mid region of the central region of the sheep MHC may be a block like structure characterized by low recombination similar to those that have been widely described in the human and mouse genomes. The discoveries reported in this thesis provide a more accurate and detailed description of the central region of the sheep MHC together with a panel of SNPs, which reflect the diversity of this important genomic region which is known to be associated with immune responsiveness. The description, for the first time, of central region haplotypes provides a practical means of seeking candidate loci associated with disease resistance and productivity traits. The application of molecular techniques will enhance the rate at which the genomic composition of this region is elucidated and the work described in this thesis will contribute to final characterization of this important complex in health and disease.

Arctic charr is the most northerly distributed salmonid and the most abundant fish in high latitude postglacial lakes. Arctic charr lives in oligotrophic water bodies where it has been able to adapt and thrive due, in part, its noted outstanding phenotypic plasticity. Throughout its geographic range, the Arctic charr has had to specialize to get the most of each ecosystem, to the point that Arctic charr were originally described as 56 different species and only later considered as many phenotypic variations of the same group, called the Arctic charr complex. With the aim of using the resources available in areas with very low primary production, Arctic charr often specialize to become different morphotypes within the same water body. Each morphotype can follow different life histories that can be anadromous or non-migratory. In several lakes, the non-migratory stocks may also differentiate further, each form with its own trophic and/or reproductive behavior. Adult sympatric forms may differ in depth distribution, size-at-maturity, time and place of spawning, color and/or other meristic characters that include differential gill raker and vertebrae numbers. The two typical forms that are found in sympatry are a small, profoundal form often termed “dwarf” Arctic charr and a large, littoral or pelagic zone resident often termed “Normal” Arctic charr. The Arctic charr colonized most of its current habitat very recently, after the ice retreat in the late Pleistocene, 10000-15000 years ago. The reproductive isolation of stocks, if it has occurred at all, occurred so recently that the accumulated genetic drift often does not yield enough data to support the genetic separation of the stocks. Since the geographic borders of the stocks tend to be unclear and because the Arctic charr is a migratory species, the management of fisheries can be difficult in light of these issues, this thesis examines the potential for identifying Arctic charr populations using Major Histocompatibility (MH) genes as molecular markers. MH genes are useful because they are not neutral markers, but are subject to selection. MH receptors present peptides to T-lymphocytes and from that interaction the immune system defines what is self or non-self and thus whether or not immune reactions should be initiated. Due to the large variety of potential pathogenic peptides to be presented, the domain of the MH receptor that binds the peptide, the peptide binding region, is the most polymorphic coding region known. Each individual has a limited number of MH alleles. Given the high degree of polymorphism in populations it is virtually impossible that two individuals will share the same set, of MHC alleles with the exception of monozygotic twins. Since MH receptors present peptides derived from pathogens, they are related to disease resistance, and some MH alleles are more effective at presenting certain peptides than others. Therefore, populations settled in a specific niche will interact with a defined variety of pathogens that will select for certain patterns in the MH alleles of the population. The selection of these MH allelic patterns occurs rapidly, since they determine the survival of the individuals during disease outbreaks. Rapid selection means that MH allelic patterns they can be used to differentiate populations that have been separated for relatively short periods of time. The MH genes of Arctic charr had not been characterized before the publication of this thesis, so the first step was their isolation and characterization. We found the MH sequences obtained to have typical characteristics of classical MH receptors, sharing similarities with other salmonids and having most of their variation in the peptide binding region. We next characterized populations of Arctic charr selected from the global distribution using the three polymorphic MH receptors. For all of the receptor we found most of the polymorphisms distributed equally amongst the populations, but the interpopulation diversity was generally enough to differentiate at least some of the studied populations. For the MH Class I we studied three non classical (UCA, UGA, UEA) and one classical (UBA) gene. For UBA and UCA we found a large degree of polymorphism while UGA and UEA were not very polymorphic. Despite the fact that the UGA gene was also not polymorphic in studies of rainbow trout, we found the gene to be the best Class I population marker for Arctic charr because it had the highest relative rates of interpopulation diversity. Thus, UGA may be exhibiting some antigen presentation functions in Arctic charr. The population analysis using MH Class II α and Class II β genes were the most successful. Particularly in the case of Class II β, the analyses arose capable of differentiating all the populations chosen for this study. Both genes showed high levels of polymorphism and high rates of non-synonymous/synonymous substitution in the exon that encodes the peptide binding region. Lastly, we used MHC Class II α and Class II β to differentiate two separate sets of morphotypes living in sympatry in Lake Kiryalta in Russia and Gander Lake in Canada. The morphotypes in Gander Lake were successfully differentiated using both MH Class II α and β allele data, while the morphotypes in Lake Kiryalta were separated only with the MH Class II β allele data. Given that the use of one or more MH genes used allowed us to differentiate the populations studied, MH genes seem to be extremely useful as population markers for Arctic charr. Since MH genes not only characterize populations according to their phylogenetic relationships, but also according to their specific adaptation to inhabited niches, we concluded that all the Arctic charr populations studied are independent evolutionary significant units of the Arctic charr species. The conclusion implies that although different stocks might be living in sympatry, they should be considered as separate species for fishery and other management purposes, because their specific adaptations to the pathogens in their ecological niche might not allow them to cross-repopulate the other stock if it were removed by over-fishing or other anthropogenic stresses.

Arctic charr is the most northerly distributed salmonid and the most abundant fish in high latitude postglacial lakes. Arctic charr lives in oligotrophic water bodies where it has been able to adapt and thrive due, in part, its noted outstanding phenotypic plasticity. Throughout its geographic range, the Arctic charr has had to specialize to get the most of each ecosystem, to the point that Arctic charr were originally described as 56 different species and only later considered as many phenotypic variations of the same group, called the Arctic charr complex. With the aim of using the resources available in areas with very low primary production, Arctic charr often specialize to become different morphotypes within the same water body. Each morphotype can follow different life histories that can be anadromous or non-migratory. In several lakes, the non-migratory stocks may also differentiate further, each form with its own trophic and/or reproductive behavior. Adult sympatric forms may differ in depth distribution, size-at-maturity, time and place of spawning, color and/or other meristic characters that include differential gill raker and vertebrae numbers. The two typical forms that are found in sympatry are a small, profoundal form often termed “dwarf” Arctic charr and a large, littoral or pelagic zone resident often termed “Normal” Arctic charr. The Arctic charr colonized most of its current habitat very recently, after the ice retreat in the late Pleistocene, 10000-15000 years ago. The reproductive isolation of stocks, if it has occurred at all, occurred so recently that the accumulated genetic drift often does not yield enough data to support the genetic separation of the stocks. Since the geographic borders of the stocks tend to be unclear and because the Arctic charr is a migratory species, the management of fisheries can be difficult in light of these issues, this thesis examines the potential for identifying Arctic charr populations using Major Histocompatibility (MH) genes as molecular markers. MH genes are useful because they are not neutral markers, but are subject to selection. MH receptors present peptides to T-lymphocytes and from that interaction the immune system defines what is self or non-self and thus whether or not immune reactions should be initiated. Due to the large variety of potential pathogenic peptides to be presented, the domain of the MH receptor that binds the peptide, the peptide binding region, is the most polymorphic coding region known. Each individual has a limited number of MH alleles. Given the high degree of polymorphism in populations it is virtually impossible that two individuals will share the same set, of MHC alleles with the exception of monozygotic twins. Since MH receptors present peptides derived from pathogens, they are related to disease resistance, and some MH alleles are more effective at presenting certain peptides than others. Therefore, populations settled in a specific niche will interact with a defined variety of pathogens that will select for certain patterns in the MH alleles of the population. The selection of these MH allelic patterns occurs rapidly, since they determine the survival of the individuals during disease outbreaks. Rapid selection means that MH allelic patterns they can be used to differentiate populations that have been separated for relatively short periods of time. The MH genes of Arctic charr had not been characterized before the publication of this thesis, so the first step was their isolation and characterization. We found the MH sequences obtained to have typical characteristics of classical MH receptors, sharing similarities with other salmonids and having most of their variation in the peptide binding region. We next characterized populations of Arctic charr selected from the global distribution using the three polymorphic MH receptors. For all of the receptor we found most of the polymorphisms distributed equally amongst the populations, but the interpopulation diversity was generally enough to differentiate at least some of the studied populations. For the MH Class I we studied three non classical (UCA, UGA, UEA) and one classical (UBA) gene. For UBA and UCA we found a large degree of polymorphism while UGA and UEA were not very polymorphic. Despite the fact that the UGA gene was also not polymorphic in studies of rainbow trout, we found the gene to be the best Class I population marker for Arctic charr because it had the highest relative rates of interpopulation diversity. Thus, UGA may be exhibiting some antigen presentation functions in Arctic charr. The population analysis using MH Class II α and Class II β genes were the most successful. Particularly in the case of Class II β, the analyses arose capable of differentiating all the populations chosen for this study. Both genes showed high levels of polymorphism and high rates of non-synonymous/synonymous substitution in the exon that encodes the peptide binding region. Lastly, we used MHC Class II α and Class II β to differentiate two separate sets of morphotypes living in sympatry in Lake Kiryalta in Russia and Gander Lake in Canada. The morphotypes in Gander Lake were successfully differentiated using both MH Class II α and β allele data, while the morphotypes in Lake Kiryalta were separated only with the MH Class II β allele data. Given that the use of one or more MH genes used allowed us to differentiate the populations studied, MH genes seem to be extremely useful as population markers for Arctic charr. Since MH genes not only characterize populations according to their phylogenetic relationships, but also according to their specific adaptation to inhabited niches, we concluded that all the Arctic charr populations studied are independent evolutionary significant units of the Arctic charr species. The conclusion implies that although different stocks might be living in sympatry, they should be considered as separate species for fishery and other management purposes, because their specific adaptations to the pathogens in their ecological niche might not allow them to cross-repopulate the other stock if it were removed by over-fishing or other anthropogenic stresses.

Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, 2002. / Includes bibliographical references. Also available on the Internet.