Thesis (PhD)--University of South Australia, 2003.

B cell chronic lymphocytic leukemia (B-CLL) is a hematologic neoplasm characterised by the proliferation and accumulation of sIgM+/D+ B cells that fail to progress to the final stages of B cell development. The malignant cells in B-CLL also express the pan-T cell antigen CD5, suggesting that CLL is a malignancy of the CD5+ subset of B cells. Additional characteristics of the malignant clone include a low proliferative index, enhanced in vivo survival and constitutive expression of the anti-apoptosis oncoprotein bcl-2. The behaviour of leukemic CD5 B cells in vitro contrasts their arrested in vivo state. That is, despite the majority of cells being arrested in the G0 phase of the cell cycle, the leukemic B cells are not irreversibly frozen as they can be induced to differentiate to Ig-secreting cells under appropriate in vitro conditions. Furthermore, leukemic CD5 B cells rapidly undergo death by apoptosis following in vitro culture. This thesis describes the requirements for in vitro activation of leukemic CD5+ B cells, the characterisation of the events involved in apoptosis of these cells as well as the identification of various growth factors capable of modulating these events. Stimulation of unfractionated peripheral blood lymphocytes (PBLs) from three patients with B-CLL with the phorbol ester PMA and the mitogens PHA and PWM resulted in significant increases in cell proliferation, RNA synthesis and 1gM secretion when compared to unstimulated cell populations. PMA was the most potent inducer of 1gM secretion and this occurred irrespective of the presence of residual T cells. PMA-induced proliferation and RNA synthesis were also independent of T cells. However, in the presence of T cells, these parameters of cellular activation were enhanced during in vitro culture. Thus, the inductive ability of PMA on leukemic CD5 B cells was independent of T cells. In contrast, activation and differentiation of the leukemic CD5 B cells into 1gM-secreting cells following culture with mitogens did not occur in the absence of T cells. Interestingly, co-stimulation of leukemic CD5+ B cells with PMA and anti-Ig induced cellular responses that exceeded those induced by either activator alone. Thus, leukemic CD5+ B cells from patients with B-CLL can be activated in vitro and differentiate in response to stimulation via both T cell-dependent and T cell-independent mechanisms. Apoptotic cell death was characterised in purified leukemic CD5 B cells obtained from six B-CLL patients. All leukemic CD5 B cell populations entered an apoptotic pathway in vitro as evidenced by a reduction in cell size, loss of cell viability and fragmentation of DNA into multimers of -180 base pairs. Following 24 hours of in vitro culture 24.0±16% of DNA was fragmented. After 8 days, the majority of DNA was fragmented, and fewer than 10% of cultured cells were viable. Examination of bcl-2 expression in the malignant B cells by flow cytometry revealed a unimodal pattern of expression in greater than 85% of cells from each B-CLL patient prior to culture. During in vitro culture, bcl-2 expression became bimodal such that the B cells displayed a bcl-2hjgh and bcl-2iow phenotype. The level of expression by the bCl2hjgh cells was similar to that observed prior to in vitro culture, indicating that bcl-2 is down-regulated in apoptosing cells. Interestingly, despite this downregulation, the overall number of cells positive for bcl-2 remained constant. This suggests that the enhanced survival of leukemic CD5+ B cells in vivo is mediated by the sustained expression of bcl-2 and that additional mechanisms exist capable of overriding the protective effect of bcl-2 when bcl-2 is present at reduced levels. Leukemic B cell apoptosis has previously been reported to be delayed or prevented by IL-4, IFN-y and IFN-a. These results were confirmed in this study where it was found that culture of leukemic CD5 B cells with IL-4 or IFN-y enhanced cell viability and delayed apoptosis in 6/6 and 5/6 populations of leukemic B cells, respectively. This function was also found to be shared by IL-2, IL-6, IL-13 and TNF-a as these cytokines enhanced cell viability and delayed apoptosis in some of the cell populations examined at a level similar to that observed for IL-4 and IFN-y. These cytokines may mediate their effect via the expression of bcl2 as culture in the presence of IL-2, IL-4, IL-6, IL-13, IFN-y or TNF-a resulted in a higher percentage of cells displaying the bcl-2high phenotype, compared to unstimulated cells. Taken together, these results suggest that autocrine and/or paracrine growth loops may play a role in the pathogenesis of B-CLL and that cytokines that prevent apoptosis in vitro may be targets for treatment of this B cell malignancy.

Type 1 Diabetes (T1D) is an autoimmune disease where the immune system destroys the insulin-producing beta cells within the pancreas. Due to the difficulty of obtaining relevant tissue samples from patients at risk of disease, many researchers have utilized the nonobese diabetic (NOD) mouse as a model of T1D due to their natural high susceptibility for this disease which shares many characteristics with human patients. This model has been critical for uncovering many mechanisms involved in the pathogenesis of T1D including the key roles played by autoreactive T cells in the destruction of beta cells. More recently, NOD mice have shown that self-reactive B cells act as important antigen presenting cells for activating and amplifying the T cell response against beta cells. In order to identify faulty self-tolerance mechanisms causing production and activation of B cells recognizing beta cell proteins, we have developed a transgenic mouse model whereby elevated numbers of B cells are made specific for a neo-self antigen whose expression is restricted to beta cells on the T1D-prone NOD genetic background and compared it to that of transgenic mice of the non-autoimmune prone C57BL/6 (B6) genetic background. These studies revealed that NOD and B6 B cells can both be effectively tolerized to the model beta cell-restricted antigen. However, provision of help from activated T cells readily overturned this tolerance on the NOD but not the B6 background. Prior evidence has associated Idd5 (chromosome 1) and Idd9/11 (chromosome 4) diabetes susceptibility loci in NOD mice with the development of self reactive B cells contributing to T1D. The gene encoding CTLA-4 has been identified as the major candidate susceptibility gene within Idd5, thus leading to our studies comparing B cell expression of this molecule in NOD and diabetes-resistant strains. Although almost always associated with down-modulating T cells responses, our studies and that of others confirm expression of CTLA-4 by activated B cells. We encountered B cell expression of CTLA-4 to vary from that of T cells, being expressed earlier and predominantly on the cell surface rather than within intracellular vesicles. Our studies also showed aberrant expression of different splice variants of CTLA-4 by NOD B cells compared to diabetes-resistant mice controlled by genes within and outside the Idd5 genetic locus. Hence, these studies raise the possibility that CTLA-4 may contribute to T1D through its actions on both T and B cells. Given the large nature of the Idd9/11 susceptibility locus in NOD mice and the absence of any strong candidate genes that may influence the diabetogenic capacity of B cells in this strain, we resorted to microarray technology to reveal putative genes within this genomic region with the potential to control the B cell phenotype. We focused our microarray studies on the first transitional (T1) B cell population in the spleen given that it is an important stage of tolerance to peripherally expressed self-antigens which have been found to possess various defects in NOD mice. Comparing gene expression profiles of NOD T1 B cells that expressed susceptibility or resistance alleles at the Idd9/11 locus identified 20 differentially expressed genes with the potential to contribute to development of diabetogenic B cells. Overall, data presented in this thesis provides a greater understanding of the molecular and cellular mechanisms underlying B cell contribution to T1D in NOD mice. These data are hoped to eventually lead to the development of selective strategies for removing or inhibiting only those B cells that contribute to development of T1D while ensuring that humoral immunity to foreign pathogens remains intact in human patients at risk of developing disease.

Although Australian epidemiology of HBV and HCV has been well described for populations groups at higher risk, but the information available for groups generally considered to be lower risk is much more limited. An understanding of the prevalence of these infections and their risk factors in antenatal women is important to guide testing policy and practice. A study was therefore conducted of the epidemiology of hepatitis B and hepatitis C infection in women. In addition, women were asked about their experience with antenatal testing. A total of 516 women participated in the survey, of these 479 (95%) women had been tested for HCV antibodies .The prevalence of HCV antibodies was 4% overall, and 2% among women who were unaware of their HCV status prior to their antenatal test. A history of injecting drug use and residing with a HCV positive person were significantly associated with HCV infection in multivariate analyses. HBV testing was conducted in 468 (99.6%) of women, and the overall prevalence was 2%. Risk factors identified were birthplace in countries of South East Asia. Women were asked about their perception of antenatal testing and pre-test information. Nearly a third (143, 30.5%) of women who had been tested for HCV infection either said that they did not know whether they had been tested, or said that they had declined testing. The corresponding proportion for HBV infection was 28.8% (135). Over 65% and 66% of women said that had not received any information about testing for HCV and HBV respectively. The finding that virtually all antenatal women were being tested for HCV was in contrast to government and non-government organisation policies of ???selective??? screening in place during the study period. Of concern was the substantial proportion of women who were tested despite reporting that they had declined their clinician???s offer to test for HCV and HBV, and the large number of women who reported an absence of pre-test information. Women who said they had received information reported the delivery and quality of information varied according to the antenatal clinician group, but perceived the overall quality as poor.

B lymphocytes constitutes an important arm of the immune system, and their response to antigen is largely dependent upon signal transduction through the B cell receptor (BCR). Such a potent receptor, however, needs to be further balanced by positive and negative regulators to prevent harmful effects that may arise from inappropriate stimulation. Src family protein tyrosine kinase Lyn is involved in both positive and negative regulation, since the both gain-of-function Lyn and loss-of-function Lyn mutations caused autoimmunity in mice. The exact signalling pathway(s) regulated by Lyn in B cells, however, are still not clear. Work presented in this thesis attempts to elucidate the biochemical mechanisms that underline the double-edged nature of Lyn in BCR signalling. (For complete abstract open document)

Group B streptococcus (GBS, S. agalactiae) and human ureaplasmas (U. parvumand U. urealyticum) are two clinically and phylogenetically related, potentialperinatal pathogens. Their relationships between genotypes and pathogenesis ofGBS and ureaplasma infection were still not well understood, one of the reason isthat both of them are still short of a very practical genotyping system. In the study,to solve the above problem we developed genotyping systems for the organisms (thesecond section). For human ureaplasmas, based on four genes/gene clusters (rRNAgene clusters, the elongation factor Tu genes, urease gene complexes and multiplebanded antigen genes), we designed many primer pairs suitable for developing species identification assays for the two newly established human ureaplasmaspecies (U. parvum and U. urealyticum). Further, based on the heterogeneity ofureaplasma multiple banded antigen gene (which contains species- and serovar-specific regions), we developed genotyping methods for each ureaplasma species.For GBS, based on three sets of molecular markers (capsular polysaccharidesynthesis gene clusters, surface protein antigen genes and mobile genetic elements),we developed a genotyping system. The primary evaluation of the genotypingsystems showed that the genotyping systems were practical alternative assays forthe conventional serotyping and they will be useful to further explore therelationships between genotypes and pathogenesis of GBS and ureaplasmainfection. In the study, we introduced novel data and tools into GBS and ureaplasmastudies especially from genomic- and bioinformatics-based molecular microbiology(the third section). For two newly established human ureaplasma species, based onthe U. parvum serovar-3 genome, and using the above four important genes/geneclusters, we exposed some interesting problems in the understanding of newureaplasma taxonomy especially in the post genomic era. For GBS, we studied thetwo published full genomes and exposed some new problems or possible future newresearch fields. In particular we found the two finished and one ongoing GBSgenomes were all non-typical and suggest that future genomic project had better have genetic population structure viewpoint. Finally, we suggested that integratedstudies of the two potential or conditional perinatal pathogens, from the viewpointof evolution, would provide a new understanding angle of the pathogenesis of thetwo organisms. Studies suggested that during coevolution, human ureaplasmas(especially U. parvum) became friendlier than their ancestors to their human host(by losing most of its virulence genes); however, GBS tried to increase its invasiveabilities (by getting more virulence genes) to fight against the human host attack.

Thesis (Ph. D.)--University of Adelaide, 1986. / Offprints of author's four journal articles in pocket. Includes bibliographical references (leaves 131-147).

Proefschrift Universiteit van Amsterdam. / Auteursnaam op omslag: Mijntje Aarts. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Universiẗat, Diss., 2006--Saarbrücken.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.