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The general aim of this project was to study aspects of the canine MHC as it relates to transplantation. The first approach, which constitutes the bulk of the thesis, involved a molecular study of canine class II major histocompatibility complex loci, known collectively as the DLA-D region, to examine the value of such analysis in determining DLA-D haplotype, and to assess its usefulness in predicting the outcome of allogeneic transplantation. A second approach targeted the molecules encoded by DLA-D loci. These studies aimed to document the kinetics of changing cell surface concentration of these molecules within transplanted tissue, to assess the potential for utilising this information in rejection diagnosis, and to attempt the immunological manipulation of the molecules as a modulatory therapy. Prior to the commencement of the work described in this thesis, no experience with DNA-based techniques existed in the laboratory at Westmead Hospital, and a series of preliminary studies was necessary to establish the range of protocols required for Southern analysis of restriction fragment length polymorphism (RFLP). The preliminary studies established that human cDNA probes derived from HLA genes can hybridise successfully to digested canine genomic DNA, that polymorphic restriction fragments can be generated with a number of restriction endonucleases, notably PstI, BglII and TaqI, and that a probe derived from HLA-DRB hybridises to the greatest number of fragments. Additional experiments indicated that a DPB probe was also useful when hybridised to PstI-digested DNA. Once the protocols for RFLP analysis were established, a systematic study was performed on DNA derived from 31 dogs that had been typed as homozygous for DLA-D region loci by mixed lymphocyte reaction. Twenty-six of these animals were from the Fred Hutchinson Cancer Research Centre, Seattle, U.S.A., and frozen cells were imported on dry ice. The other five animals were bred in experimental dog colonies at Westmead Hospital. The Seattle animals represented 13 different haplotypes, but no information was available on whether the Westmead dogs shared any of these. A total of 15 different RFLP patterns were identified in the Seattle and Westmead dogs. Eleven patterns were revealed using PstI-digested DNA hybridised to a DPB probe. The other four patterns resulted from the incorporation of data from either TaqI-, RsaI- or PvuII-digested DNA. The RFLP patterns of the Seattle type D16 and Westmead type W.02 could not be distinguished. A limited amount of material prevented a complete assessment of the Dw9 and D7 types, but analysis of TaqI-digested DNA grouped these animals with the Seattle types D8 and D16, and the Westmead type W.02. Overall the data indicated that Southern analysis is an effective means of predicting DLA-D haplotypes, at least in homozygote dogs. A sample of mixed-breed dogs was studied to assess the general applicability of the RFLP-based typing system. RFLP patterns generated with the enzymes PstI, RsaI and TaqI, and hybridised to either DPB or DRB probes, could not provide enough information to assign a definitive genotype in these animals. The failure of the system in this case resulted from an insufficient number of restriction fragments that were unique to one of the defined haplotypes. However, the data indicated that the degree of polymorphism detected by a DPB probe in PstI-digested DNA from mixed-breed animals would be useful in transplantation studies for predicting MHC non-identity between tissue donors and recipients. |
