Master of Science / Periodontal disease is one of the most widespread diseases in humans and is characterised by chronic gingival inflammation and B cell accumulation and resorption of the crest of alveolar bone with subsequent loss of teeth. Porphyromonas gingivalis has been identified as a putative aetiological agent for periodontitis. The aim of the research presented in this thesis was to investigate, using in vitro systems, the responses of autoreactive B-1 and B-2 cells to enterobacterial and nonenterobacterial lipopolysaccharide (LPS) to shed light on the pathogenesis of chronic periodontitis and other diseases involving B cell accumulation and autoantibody production. The hypotheses tested were: (1) B cells respond differently to enterobacterial and non-enterobacterial LPS. (2) B-1 cells are activated by a lower concentration of LPS than B-2 cells. (3) LPS stimulation results in preferential accumulation of B-1 cells. Findings consistent with these hypotheses would provide new evidence for different roles for B-1 and B-2 cells in immune responses and that LPS stimulation could lead to B-1 cell accumulation in diseases thus characterised. Initial experiments investigated the responses of representative B-1 (CH12) and B-2 (WEHI-279) cell lines to preparations of P. gingivalis and Salmonella enteritidis LPS utilising flow cytometric and quantitative molecular methods. The cell lines responded differently to the two LPS preparations. There were significant but limited effects on viability and proliferation in the WEHI-279 cell line, but no significant changes in mRNA expression levels for genes including Toll-like receptors (TLR2, TLR4, RP105), immunoglobulin (IgM), cytokines (IL-6, IL-10), co-stimulatory molecules (CD80, CD86), and regulators of apoptosis (Bcl-2, Bax). In the CH12 cell line however, LPS stimulation had greater effect. Addition of S. enteritidis LPS from a threshold level of 100ng/mL was found to rescue the cells from death, reflected by the percentage viability and proliferation. Stimulation of CH12 cells with S. enteritidis LPS also led to a decrease in expression of RP105 mRNA, which may be part of a negative feedback loop. Interestingly, stimulation with low concentrations P. gingivalis LPS appeared to inhibit proliferation but high LPS concentrations stimulated proliferation of CH12 cells, although no further significant effects were noted in other analyses. Evidence was found that CH12 cells have a high basal level of activation. This suggests that this line is constitutively activated. Stimulation with P. gingivalis or S. enteritidis LPS did not affect the level of CD80 mRNA expression. It is possible that the CH12 line constitutively expresses a maximal level of CD80 (and possibly CD86) and further stimulation will not cause any increase. Since S. enteritidis LPS appeared to have more pronounced effects on both B cell populations, this LPS was used to further investigate B cell subset responses in a mixed splenocyte culture system. Experiments examining percentage viability and number of viable cells indicated that B-1 and B-2 B cells responded differently to LPS stimulation. A threshold level for B-2 cell response (significant increase in cell number) was found to be 100ng/mL LPS, in contrast to the B-1 B cell subset which were only significantly different to the unstimulated cells when stimulated with 50μg/mL LPS. By examining the expression of CD80, the majority of murine splenic B-1 cells were found to activated prior to any LPS stimulation in vitro. In contrast, the B-2 subset showed significant increase in CD80 expression only at high (≥10μg/mL) LPS concentrations. Studies of the division index of B-1 and B-2 cells showed a significant response in both subsets following stimulation with 1μg/mL and 10μg/mL LPS. However, overall, the results are inconsistent with LPS driving the preferential accumulation of B-1 cells in disease states. These experiments provided useful evidence that supported the idea that B-1 and B-2 cells respond differently to LPS. However, these studies were unable to directly address the role of P. gingivalis LPS in periodontitis. It may be that P. gingivalis LPS could have different effects to S. enteritidis LPS on primary B cells. It is still possible that B-1 cells may be more sensitive to P. gingivalis, as opposed to S. enteritidis LPS. Studies by other groups have suggested that the TH1/TH2 profile is skewed towards TH2 in chronic periodontitis and that P. gingivalis may drive this shift via its ability to signal through TLR2 (and modulate TLR4 signalling). Further, recent studies in our laboratories have found that P. gingivalis gingipains are able to polyclonally activate B cells and to break down both IFNγ and IL-12. Future studies should further examine the effects of B-1 and B-2 interactions in the mixed lymphocyte system together with subsequent studies utilising human periodontitis biopsies. The results presented in this thesis, together with work undertaken by other investigators, suggests that LPS could perturb the normal homeostatic mechanisms of the B-1 B cell-subset and increase polyclonal activation therefore contributing to the genesis of pathologies such as chronic periodontitis.

The human hepatitis B virus (HBV) is a small hepatotropic virus, which affects approximately 350 million chronic sufferers worldwide. It has a compact 3.2 kbp dsDNA genome encoding four major overlapping genes namely core, polymerase, surface and X required for its replication. The virus synthesises a pregenomic RNA (pgRNA) which functions both as an RNA intermediate for reverse transcription into the DNA genome and as the mRNA for the translation of the core (C) and polymerase (P) proteins. The core overlaps the polymerase gene and is translated at a 10 to 1 ratio. The polymerase gene translated from the P AUG codon is preceded by at least 4 upstream AUG codons (uAUGs), namely C AUG, C1 AUG, J AUG and C2 AUG. Various mechanisms have been implicated in the synthesis of the polymerase protein. This led to the currently accepted model which involves leaky scanning and a reinitiation mechanism in polymerase synthesis. However, multiple sequence alignment of the pgRNA revealed a short upstream open reading frame (uORF) highly conserved at the nucleotide level in all HBV subtypes and mammalian hepadnaviruses. This previously unreported uORF, designated as C0 ORF in this study is also conserved in its position and length. Past studies have either omitted this uORF in their test constructs or ignored its potential role. The C0 ORF has a conserved weak initiation context and is located within the epsilon structure within the 5' leader of the pgRNA, required for viral encapsidation. Importantly, the C0 ORF precedes and overlaps the core ORF, which may suggest an alternative model in which the core and polymerase may be translated and coordinately regulated. Fusion of the C0 ORF to luciferase showed for the first time that this uORF is translated through the detection of reporter activity (~20% of C) and also visualisation of the fusion protein via western analysis using anti-C0 and anti-luciferase antibodies. Subsequent removal of the C0 ORF implicated a role in repressing downstream core fusion protein synthesis in HepG2 cells. A similar repression was observed on J expression. To study the effect of C0 on downstream polymerase translation, a pgRNA-like DNA construct was made and subsequent mutations introduced. Mutation of the C0 AUG led to an increase in initiation at the downstream P AUG. Alteration of the existing weak initiation context to an optimal context which favours stronger initiation consistently showed a potential role for C0 ORF in facilitating reinitiation at certain downstream initiation codons including P AUG. Mutations of other uAUGs preceding the P AUG were also done to better understand their roles in regulating polymerase synthesis. The removal of the C AUG markedly increased expression from the P AUG. This study revealed other internal uAUGs in-frame to the C AUG, namely the C1 and C2 AUGs are also effectively translated, further reducing availability of translating ribosomes to downstream P AUG. Indeed the removal of the C1 and C2 AUGs led to a corresponding increase in initiation from the P AUG. Initiation at the internal J AUG was also reported and its removal showed a significant decrease in expression from the P AUG, consistent with the previous model implicating reinitiation at the P initiation site after translation of the short J ORF. The inhibitory role of the 5 uAUGs prior to the P AUG were confirmed when all were removed, giving rise to translation almost equal to that at C AUG. Taken together, these results suggest a new model in which the HBV C0 ORF plays a key role in controlling core and polymerase synthesis by repressing core translation and making available more ribosomes to downstream AUGs possibly facilitating translation reinitiation. In addition, the translation of the C0 ORF across the [epsilon] region may also preclude encapsidation, potentially acting as a switch discriminating the pgRNA template between encapsidation and translation. Therefore, the highly conserved [epsilon] region and C0 ORF present an excellent target for molecular based antiviral drugs (antisense oligonucleotides, aptamers, ribozymes) potentially providing new anti HBV drugs.

The human hepatitis B virus (HBV) is a small hepatotropic virus, which affects approximately 350 million chronic sufferers worldwide. It has a compact 3.2 kbp dsDNA genome encoding four major overlapping genes namely core, polymerase, surface and X required for its replication. The virus synthesises a pregenomic RNA (pgRNA) which functions both as an RNA intermediate for reverse transcription into the DNA genome and as the mRNA for the translation of the core (C) and polymerase (P) proteins. The core overlaps the polymerase gene and is translated at a 10 to 1 ratio. The polymerase gene translated from the P AUG codon is preceded by at least 4 upstream AUG codons (uAUGs), namely C AUG, C1 AUG, J AUG and C2 AUG. Various mechanisms have been implicated in the synthesis of the polymerase protein. This led to the currently accepted model which involves leaky scanning and a reinitiation mechanism in polymerase synthesis. However, multiple sequence alignment of the pgRNA revealed a short upstream open reading frame (uORF) highly conserved at the nucleotide level in all HBV subtypes and mammalian hepadnaviruses. This previously unreported uORF, designated as C0 ORF in this study is also conserved in its position and length. Past studies have either omitted this uORF in their test constructs or ignored its potential role. The C0 ORF has a conserved weak initiation context and is located within the epsilon structure within the 5� leader of the pgRNA, required for viral encapsidation. Importantly, the C0 ORF precedes and overlaps the core ORF, which may suggest an alternative model in which the core and polymerase may be translated and coordinately regulated. Fusion of the C0 ORF to luciferase showed for the first time that this uORF is translated through the detection of reporter activity (~20% of C) and also visualisation of the fusion protein via western analysis using anti-C0 and anti-luciferase antibodies. Subsequent removal of the C0 ORF implicated a role in repressing downstream core fusion protein synthesis in HepG2 cells. A similar repression was observed on J expression. To study the effect of C0 on downstream polymerase translation, a pgRNA-like DNA construct was made and subsequent mutations introduced. Mutation of the C0 AUG led to an increase in initiation at the downstream P AUG. Alteration of the existing weak initiation context to an optimal context which favours stronger initiation consistently showed a potential role for C0 ORF in facilitating reinitiation at certain downstream initiation codons including P AUG. Mutations of other uAUGs preceding the P AUG were also done to better understand their roles in regulating polymerase synthesis. The removal of the C AUG markedly increased expression from the P AUG. This study revealed other internal uAUGs in-frame to the C AUG, namely the C1 and C2 AUGs are also effectively translated, further reducing availability of translating ribosomes to downstream P AUG. Indeed the removal of the C1 and C2 AUGs led to a corresponding increase in initiation from the P AUG. Initiation at the internal J AUG was also reported and its removal showed a significant decrease in expression from the P AUG, consistent with the previous model implicating reinitiation at the P initiation site after translation of the short J ORF. The inhibitory role of the 5 uAUGs prior to the P AUG were confirmed when all were removed, giving rise to translation almost equal to that at C AUG. Taken together, these results suggest a new model in which the HBV C0 ORF plays a key role in controlling core and polymerase synthesis by repressing core translation and making available more ribosomes to downstream AUGs possibly facilitating translation reinitiation. In addition, the translation of the C0 ORF across the [epsilon] region may also preclude encapsidation, potentially acting as a switch discriminating the pgRNA template between encapsidation and translation. Therefore, the highly conserved [epsilon] region and C0 ORF present an excellent target for molecular based antiviral drugs (antisense oligonucleotides, aptamers, ribozymes) potentially providing new anti HBV drugs.

Thesis (D. Min.)--Harding Graduate School of Religion, 1987. / Includes bibliographical references (leaves 240-254).

The homeodomain-interacting protein kinase (HIPK) family is comprised of four evolutionarily conserved and highly related serine/threonine kinases originally identified as co-repressors for homeodomain-containing transcription factors. While the HIPKs are most noted for regulation of apoptosis, proliferation and differentiation, I report a pleiotropic function of HIPK1 within the B cell lineage. Although lymphocyte development was normal within the thymus and bone marrow of HIPK1-deficient (HIPK1-/-) mice, the spleen exhibited a reduced number of transitional and follicular (FO) B cells, but with an increase in the marginal zone (MZ) B cell population. HIPK1-/- B cells exhibited impaired proliferation in response to B cell receptor (BCR) cross-linking in vitro; and immunization of HIPK1-/- mice with T-independent type 2 (TI-2) antigen resulted in a significantly impaired humoral response despite the expanded MZ B cell population. Immunization with T-dependent (TD) antigen resulted in a kinetically delayed response, with impaired affinity maturation. Identification of a kinase-substrate interaction between HIPK1 and the B cell adaptor 3BP2 suggests a potential context for HIPK1 function in BCR signaling. HIPK1-/- B cells were uniquely resistant to reactive oxygen species (ROS)-induced apoptosis, but equally susceptible to UV- and γ-irradiation compared to controls. In vitro class-switch recombination (CSR) assays revealed that HIPK1 is required for the negative regulation of CSR. HIPK1-/- B cell cultures harbored more viable cells, more switched cells, and elevated AID mRNA levels. The findings presented in this thesis demonstrate that HIPK1 is required for splenic B cell homeostasis and optimal BCR-responsiveness. In contrast, HIPK1 is also required for the negative regulation of CSR, possibly by mediating CSR-induced apoptosis.

Graduation date: 1994

Thesis (M.A.)--Ohio State University. / Includes bibliographical references (leaves 119-128). Available online via OhioLINK's ETD Center

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Thesis (M. Phil.)--University of Hong Kong, 1976. / Also available in print format.

L’Ins(1,3,4,5)P4 produit par l’Ins(1,4,5)P3 3-kinase de type B (Itpkb) est nécessaire au développement des thymocytes et lymphocytes T murins. Trois hypothèses sont admises quant à la fonction physiologique et au mécanisme d’action de cet inositol phosphorylé : la première postule que l’Ins(1,3,4,5)P4 module la réponse calcique intracellulaire ; la seconde, que cet inositolphosphate est un intermédiaire métabolique dans la synthèse d’inositols plus hautement phosphorylés ; la dernière, que l’Ins(1,3,4,5)P4 module la localisation subcellulaire et la fonction de protéines capables de la reconnaître par des domaines spécifiques de liaison. Afin d’investiguer cette dernière hypothèse, nous avons analysé la physiologie des lymphocytes B invalidés pour Itpkb et avons généré et analysé des souris transgéniques d’addition pour Rasa3, récepteur potentiel à l’Ins(1,3,4,5)P4. Les lymphocytes B déficients en Itpkb présentent un défaut de survie car ils ne peuvent activer correctement les protéines kinases Erk1/2 suite à la stimulation du BCR de surface. Cela conduit à la surexpression anormale de la protéine pro-apoptotique Bim. La diminution de l’expression de Bim est suffisante dans ce modèle pour restaurer une fonction normale des lymphocytes B. In vitro, Nous avons montré que l’Ins(1,3,4,5)P4 est nécessaire à la translocation de Rasa3, protéine favorisant l’inactivation de la voie de Ras, de la membrane vers le cytoplasme. L’étude de lymphocytes invalidés pour Itpkb dans un modèle de BCR transgénique semble montrer que des anomalies de réponse calcique ne participent pas au phénotype. En conclusion, nos résultats indiquent qu’une des voies de signalisation préférentielle de l’Ins(1,3,4,5)P4 passe par la modulation de la localisation subcellulaire de protéines possédant un domaine d’affinité pour l’Ins(1,3,4,5)P4 telle que Rasa3.