No description available.

No description available.

No description available.

No description available.

No description available.

PI3Ks (phosphoinositide 3-kinases), a family of enzymes expressed in immune cells, are activated in response to a wide variety of stimuli by generating second lipid messengers. A subset of singnaling molecules containing lipid-binding pleckstrin homology (PH) domains are downstream molecules of PI3K signaling pathway, essential to mediate the functional outcomes of PI3Ks. Bam32 / DAPP1 is a PH domain-containing adaptor protein, which was discovered from human tonsil germinal centers (GCs); however, its biological function related to GCs, where efficient T-cell-dependent (TD) antibody responses are generated, is unknown. This thesis is focused on the effect of genetic or pharmacological blockade of PI3K p110delta activity on T and B cells, and the role of Bam32 in GC responses. Type 2 cytokine responses are significantly decreased in p110delta-inactivated mice, whereas Type 1 cytokine responses are increased or comparable after primary and secondary immunization. Hallmarks of asthma, airway inflammation and respiratory hyper-responsiveness are dramatically reduced in those mice. Adoptive transfer of OVA-primed splenocytes from normal, but not p110delta-inactivated mice could induce airway eosinophilia in naïve, airway-challenged recipient mice. These data demonstrate a novel functional role for p110delta signaling in induction of Type 2 responses in vivo and may offer a new therapeutic target for Th2-mediated airway disease. Paradoxically, serum IgE levels are markedly increased in OVA-immunized p110delta-inactivated mice despite lower level of swich factor IL-4. In vitro studies showed that p110delta is required to restrain IgE class switch recombination in a B-cell intrinsic manner. Blockade of PI3K activity using broad-spectrum PI3K inhibitors PIK-90 and PI-103 generates similar results. In vivo administration of p110delta-selective inhibitor IC87114 into OVA-immunized mice results in selective elevation of antigen-specific IgE production. Disruption of p110delta signaling leads to increased germline transcription at the epsilon locus (epsilon GLT) and increased induction of activation induced cytidine deaminase (AID) enzyme, suggesting deregulation at the level of the isotype switch process. Moreover, p110delta signaling selectively regulates the expression level of transcription factor Bcl6 and IRF4, which may be responsible for the regulation of AID and epsilon GLT. PI3K signaling regulates multiple steps of GC development, and Bam32 may be involved. GCs dissipate prematurely in Bam32-deficient mice after immunization with OVA/alum. In vitro, Bam32-deficient B cells are functional competent in proliferation, chemotaxis, isotype switching and plasma cell differentiation in response to signals present in GCs. In vivo, Bam32-deficient GC B cells proliferate normally; however, they are more apoptotic. Adoptive transfer studies indicated that intrinsic defect of Bam32-/- B cells leads to premature GC dissolution. Additionally, GCs formed by Bam32-/- B cells contain fewer T cells, implying that Bam32 is required for B cell-dependant T cell accumulation within established GCs. Treatment of Bam32-/- mice with agonistic anti-CD40 fully restored GC persistence and IgG1 isotype switching, demonstrating that Bam32-deficient GC B cells are functionally competent when access to cognate signals is not limiting. Collectively, those data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival. In general, by focusing on PI3K p110delta and its downstream adaptor protein Bam32, my studies clearly indicate that p110delta is a potential therapeutic target for the treatment of Th2-induced airway inflammation. The unexpected immunomodulatory acitivity on IgE switching associated with multiple PI3K inhibitor compounds is first discovered in this thesis, suggesting that more need to be investigated in this aspect before those inhibitor compounds are widely used in the clinic. Furthermore, the specific regulatory role of Bam32 in GCs represents a unique model for us to study the late GC checkpoint in regarding to in vivo GC B cell and T cell interaction, which is an important issue need to be clarified in order to fully understand GC responses.

PI3Ks (phosphoinositide 3-kinases), a family of enzymes expressed in immune cells, are activated in response to a wide variety of stimuli by generating second lipid messengers. A subset of singnaling molecules containing lipid-binding pleckstrin homology (PH) domains are downstream molecules of PI3K signaling pathway, essential to mediate the functional outcomes of PI3Ks. Bam32 / DAPP1 is a PH domain-containing adaptor protein, which was discovered from human tonsil germinal centers (GCs); however, its biological function related to GCs, where efficient T-cell-dependent (TD) antibody responses are generated, is unknown. This thesis is focused on the effect of genetic or pharmacological blockade of PI3K p110delta activity on T and B cells, and the role of Bam32 in GC responses. Type 2 cytokine responses are significantly decreased in p110delta-inactivated mice, whereas Type 1 cytokine responses are increased or comparable after primary and secondary immunization. Hallmarks of asthma, airway inflammation and respiratory hyper-responsiveness are dramatically reduced in those mice. Adoptive transfer of OVA-primed splenocytes from normal, but not p110delta-inactivated mice could induce airway eosinophilia in naïve, airway-challenged recipient mice. These data demonstrate a novel functional role for p110delta signaling in induction of Type 2 responses in vivo and may offer a new therapeutic target for Th2-mediated airway disease. Paradoxically, serum IgE levels are markedly increased in OVA-immunized p110delta-inactivated mice despite lower level of swich factor IL-4. In vitro studies showed that p110delta is required to restrain IgE class switch recombination in a B-cell intrinsic manner. Blockade of PI3K activity using broad-spectrum PI3K inhibitors PIK-90 and PI-103 generates similar results. In vivo administration of p110delta-selective inhibitor IC87114 into OVA-immunized mice results in selective elevation of antigen-specific IgE production. Disruption of p110delta signaling leads to increased germline transcription at the epsilon locus (epsilon GLT) and increased induction of activation induced cytidine deaminase (AID) enzyme, suggesting deregulation at the level of the isotype switch process. Moreover, p110delta signaling selectively regulates the expression level of transcription factor Bcl6 and IRF4, which may be responsible for the regulation of AID and epsilon GLT. PI3K signaling regulates multiple steps of GC development, and Bam32 may be involved. GCs dissipate prematurely in Bam32-deficient mice after immunization with OVA/alum. In vitro, Bam32-deficient B cells are functional competent in proliferation, chemotaxis, isotype switching and plasma cell differentiation in response to signals present in GCs. In vivo, Bam32-deficient GC B cells proliferate normally; however, they are more apoptotic. Adoptive transfer studies indicated that intrinsic defect of Bam32-/- B cells leads to premature GC dissolution. Additionally, GCs formed by Bam32-/- B cells contain fewer T cells, implying that Bam32 is required for B cell-dependant T cell accumulation within established GCs. Treatment of Bam32-/- mice with agonistic anti-CD40 fully restored GC persistence and IgG1 isotype switching, demonstrating that Bam32-deficient GC B cells are functionally competent when access to cognate signals is not limiting. Collectively, those data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival. In general, by focusing on PI3K p110delta and its downstream adaptor protein Bam32, my studies clearly indicate that p110delta is a potential therapeutic target for the treatment of Th2-induced airway inflammation. The unexpected immunomodulatory acitivity on IgE switching associated with multiple PI3K inhibitor compounds is first discovered in this thesis, suggesting that more need to be investigated in this aspect before those inhibitor compounds are widely used in the clinic. Furthermore, the specific regulatory role of Bam32 in GCs represents a unique model for us to study the late GC checkpoint in regarding to in vivo GC B cell and T cell interaction, which is an important issue need to be clarified in order to fully understand GC responses.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

Immune responses are presumed to contribute to host fitness, either by fighting off infections or via immunopathology. Research in this thesis sought to relate the magnitude of a putative immune response to infection and host and parasite fitness. The experiments and field studies presented here all focus on the interactions between the freshwater crustacean, Daphnia magna and its sterilizing bacterial endoparasite, Pasteuria ramosa, using the number of circulating haemocytes as a measure of host immune activity. I found substantial genetic variation in Daphnia’s cellular response to P. ramosa, and that Daphnia genotypes that mount the strongest cellular responses are the most likely to get infected and suffer sterilization. Thus, a strong cellular response is associated with low, as opposed to high host fitness potential. There were also some host genotypes that mounted a weaker cellular response and did not go on to suffer infection, and some that lacked a cellular response and also never suffered infection with P. ramosa. These findings led to a heuristic two-stage model for infection, where the parasite has to (1) pass from the host gut to haemolymph and then (2) successfully overcome haemolymph-based immune effectors to reproduce and achieve fitness. I also demonstrate that both the magnitude of host cellular response and likelihood of infection increases with initial parasite dose in susceptible host genotypes, and that host cellular response is associated with likely infection under both host and parasite genetic variation. Parasitised Daphnia also have substantially more circulating haemocytes than their healthy counterparts in both the laboratory and in the wild, where there is substantial genetic and environmental variation. This is one of the very few examples of how an immune response designates low host and high parasite fitness potential in a wild system. Finally, using a mixture of field study and common garden experiment, I demonstrate evolution in parasite infection traits over the course of an epidemic in a wild population, and that this evolution is associated with a decline in host abundance.

Despite the availability of live attenuated measles virus vaccines, a large number of measles-associated deaths occur among infants in developing countries during the "window of susceptibility" (age 4-9 months). During this period declining maternal antibody titers are no longer protective against wild-type measles virus (MV) and impede successful immunization with the live attenuated vaccines. Therefore, the development of a safe vaccine that would induce protective immunity in the presence of maternally derived MV-specific antibodies in young infants and would close the "window of susceptibility" is desirable. Since adenoviruses have been shown as suitable vaccine candidates capable of eliciting potent protection against mucosal infectious diseases, the ability of an adenovirus-vectored anti-measles vaccine to elicit robust immune responses against MV was assessed in this study. Mice immunized intramuscularly or intranasally with a combination of human adenovirus serotype 5 (Ad5) recombinants expressing MV hemagglutinin (H) and fusion (F) glycoproteins developed MV-specific neutralizing antibody titers similar for both routes of immunization. However, intramuscular immunization of mice with Ad5 recombinants resulted in induction of a predominant T helper type (Th1) immune response, whereas intranasal immunization induced a balanced Th1/Th2 immune response. Furthermore, intranasal immunization resulted in increased titers of MV-specific immunoglobulin A (IgA) in lungs in comparison to intramuscularly immunized animals. The ability of the Ad5 recombinants to induce protective immune responses in cotton rats by different routes of administration was also evaluated. Cotton rats that received a single dose of the Ad5 recombinants intramuscularly or intranasally experienced a rise in MV-specific neutralizing antibody titers and reduction of the viral RNA load in the lung tissue after intranasal MV challenge. In addition, the largest reduction in viral replication was observed in the group of cotton rats inoculated with the Ad5 recombinants intranasally. Based on these observations, the Ad5-based vaccine appears to be a suitable candidate against measles. Furthermore, a capability of purified globular head domain of MV H protein produced in a human cell line to induce MV-specific immune responses in mice was tested. Subcutaneous immunization of mice with the recombinant protein alone resulted in both humoral and cell-mediated immunity, characterized by the production of MV-specific serum IgG and neutralizing antibodies, as well as interferon-gamma and interleukin 5 (IL-5) production by in vitro restimulated splenocytes. The former responses were further enhanced by formulation of the protein with aluminium hydroxide. However, very low numbers of INF-gamma secreting cells and low levels of IgG2a in the serum suggested a Th2 immune response. Novel adjuvants (Th1-directing), as well as MV F protein should be considered for the inclusion into the vaccine formulations to induce more balanced Th1/Th2 immune responses against measles.