Typescript (photocopy) / xiii, 192 leaves, [44] leaves of plates : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--Dept. of Medicine, University of Adelaide, 1980

Using intact Leishmania donovani promastigotes or purified L. donovani lipophosphoglycan (LPG) as immunogens, four LPG-specific monoclonal antibodies (mAbs) were derived. Two of these mAbs (CA7AE and BF9CC) specifically bound to an epitope consisting of the repeating phosphorylated Gal-(β1,4)-Man disaccharide portion of the LPG molecule. MAb CA7AE also bound antigens in L. donovani promastigoteconditioned culture medium; specifically, the secreted forms of LPG (phosphoglycan) and acid phosphatase, demonstrating that major secreted glycoconjugates of L. donovani share phosphorylated carbohydrate epitope(s). The two other mAbs (L157 and L98) bound to a parasite-derived protein component that was discovered to be tightly associated with the phosphocarbohydrate core region of the LPG molecule (LPG-associated protein; LPGAP). These are the first defined epitopes of LPG. Immunochemical assays were used to analyze the distribution of the LPG repeat and LPGAP epitopes over a wide sampling of Leishmania species and strains. MAb CA7AE recognized, to varying extents, epitopes from most of the Leishmania species examined; both as parasite surface-exposed, membrane-bound molecules and as antigens released into parasite-conditioned culture medium. The anti-LPGAP mAbs bound to all twenty Leishmania and Trypanosoma strains assayed but not to the surface of living parasites. None of the anti-LPG mAbs bound the amastigote form of the parasites. Experiments involving amastigote-to-promastigote in vitro transformation showed that the CA7AE epitope was expressed on the surface of transforming cells within 5 hours of culture at 26°C. The epitope was excreted into the culture supernatant within 15 hours. In addition, the mAb CA7AE “epitope was detected in 50% of sera tested from L. donovani infected (Kala-azar-positive) patients. Murine macrophages, infected with L. donovani promastigotes, were examined by immunofluorescence for the expression of LPG epitopes. The CA7AE epitope, detected as early as 5-10 minutes post infection (p.i.), was initially localized to the immediate area of internalization of the promastigote into the macrophage with even distribution over the entire macrophage surface by 25 minutes p.i. These epitopes remained on the macrophage surface until approximately 88 hours p.i Acetone permeabilization of the macrophages, prior to mAb probing, exposed LPG epitopes present within the macrophages to at least 160 hours p.i. Treatments which inhibited macrophage phagolysosomal degradation processes had no effect on epitope expression whereas reagents that affected macrophage membrane flow, and thus phagocytosis, drastically reduced or abolished expression. Purified LPG or de-lipidated LPG were also shown to bind to a variety of different cell types but in a temperature-independent manner. The early and continued expression of LPG epitopes on the macrophage surface suggests the possibility that LPG epitopes may be involved in the immune response which is directed to Leishmania-infected macrophages. Lymph node cells from mice primed with L. donovani LPG or LPGAP or with living, virulent L. donovani promastigotes were specifically stimulated to proliferate in vitro by the LPGAP moiety of the LPG molecule. The T cell response was antigen-specific, dose-dependent, and non-mitogenic. In addition, purified T lymphocytes primed with purified LPG or LPGAP were not stimulated by LPG or LPGAP in vitro unless promastigote-infected or LPG-pulsed or LPGAP-pulsed macrophages were added. Recognition of LPGAP epitopes was an MHC-restricted event. LPGAP epitopes specifically stimulated CD4+CD8-, IL-2 secreting T lymphocytes and that active antigen processing by macrophages was required for T cell stimulation. Both L. donovani LPG and L. tropica LPG which have antigenically different repeat epitopes but which share LPGAP epitopes stimulated lymphocyte proliferation independent of the LPG source used for priming. The T cell stimulation caused by LPGAP was not species-specific and since the responding T cells were of the Th 1 phenotype and recognized epitopes in amastigotes, the LPGAP epitopes are very likely of considerable importance in Leisimania-specific immunity. The data suggests that Leishmania LPGAP is a potential vaccine candidate for leishmaniasis. / Graduate

No description available.

I investigated the humoral immune response to streptococcal cell walls (SCW) in arthritis susceptible Lewis and resistant Fisher rats. All rats were given a single intraperitoneal injection of either SCW or saline (controls). Rats were sacrificed, three rats per time point, over an eleven week period and serum was collected for ELISA. SCW injected Lewis rats produced anti-SCW antibody, whereas control rats did not. Anti-SCW antibody was significantly elevated over controls between days 14-28 (post injection). Both saline and SCW injected Fisher rats produced anti-SCW antibody, but with different kinetics. Anti-SCW antibody increased by day 7 and remained elevated over controls till day 21, after which there was no difference. ELISA were designed to determine the SCW epitope(s) recognized by anti-SCW antibody. Formamide extracts of SCW, peptidoglycan and polysaccharide, were investigated along with the terminal epitope of polysaccharide, N-acetyl-D-glucosamine, and the peptidoglycan precursor peptide. The data revealed that anti-SCW antibody was directed against a combined SCW epitope, given the lack of significant binding to any of the SCW epitopes tested. Isotype analysis of anti-SCW antibody revealed that the Lewis response was composed primarily of IgG2a whereas the Fisher response was composed primarily of IgM. Binding of rat IgG isotypes to whole streptococcus, SCW, peptidoglycan, and polysaccharide was investigated, given the possibility of background binding by the streptococcal Fc-receptor. Streptococcal binding of rat IgG was specific for IgG2c and the polysaccharide portion of SCW was necessary for binding. Passive immunization of naive Lewis rats with antibody from rats with active arthritis was ineffective at transferring the disease. However, subcutaneous injection of affinity purified anti-SCW antibody or IgG into Lewis rats, followed twenty-four hours later by a single intraperitoneal injection of SCW, suppressed the acute phase and inhibited the chronic disease. IgM rheumatoid factor (RF) was present in the serum of both saline and SCW injected Lewis and Fisher rats. However, SCW injection only induced a significant increase in IgM RF (between days 3-7) in Lewis rats. Passive immunization of Fisher rats with affinity purified IgM RF (from Lewis serum), three days post SCW injection, was ineffective at inducing arthritis.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Vaccination against Marek's disease (MD) is greatly enhanced by host genetic resistance. Genes of the growth hormone (GH) axis have been reported to affect the ontogeny and effector functions of cells of the immune system. Two strains of White Leghorn chickens bred for contrasting homozygous markers in the GH and GH receptor (GHR) genes were challenged with MDV. Contrasts for the significant interaction between marker genotype and tissue indicated that the GH/GHR marker genotype caused a shift in the distribution of the viral load in lymphoid tissues in the two strains. The analysis suggests that genetic variations in genes of the GH axis may differentially affect the host response to MDV replication in lymphoid tissues. Regarding the early time course of infection, at day 6 the viral load was highest in the thymus, while at day 10, it was highest in the spleen, indicating that the virus may have accumulated in the spleen or was continuing to replicate in this tissue. (Abstract shortened by UMI.)

Marek's disease (MD) is a highly contagious and economically important disease in the poultry industry. It is caused by an oncogenic avian herpes virus. The ability of the virus to evolve into new strains is a continual threat. Vaccination, proper management and genetic resistance are required to completely eliminate the pathogen. The discovery of several markers associated with MD resistance shows that genetic selection for resistance is feasible. Our objective was to identify markers in QTL regions that are associated with MD viraemia. The markers analysed were in the ODC gene, the GH gene and two chemokine genes, all of which are candidate genes for immune responsiveness. A database in a commercial strain of White Leghorn chickens was created. Heterozygous males and homozygous females were identified. The offspring were challenged with MD virus and spleen and thymus samples were collected six days after infection. The viral titre was quantified using competitive PCR. The data was analysed using non-parametric statistics. We found that the paternal alleles of a Hindlll RFLP in the ODC gene were associated with differences in MD viraemia in one of the six sire families analysed. In addition, a Sacl RFLP located in the GH gene also segregated for alleles, which affected MD viraemia. The analysis of the ODC gene was extended to include a second RFLP at a Msp\ site. Together with the Hindlll RFLP it defines three different haplotypes. One genotypic class AB (Hindlll (+/-), Mspl (+/+)) was associated with low vireamia in the thymus and the genotype BB (Hindlll (-/-), Mspl (+/+)) with high viraemia in the spleen. The result suggests that genetic variations in the ODC and GH gene affect MD viraemia. However, we cannot exclude that the observed effects might be due to linkage disequilibrium with adjacent genes. In the latter case, chromosome 3 and chromosome 1, which harbour the ODC and GH gene respectively, must segregate for regions that affect viraemia. The markers identified in this analysis can be used in marker assisted selection.

No description available.

No description available.