William Sha - US grants

DESCRIPTION (Adapted from the Investigator's abstract): NF-kappaB/Rel transcription factors are a family of five proteins which function as dimer complexes in the regulation of genes involved in innate and specific immunity. This study will examine how these transcription factors regulate in vivo immune responses. Our experimental approach will utilize gene-targeted mice for individual family members in combination with two unique experimental approaches. The first approach uses a histochemical marker that directly monitors in vivo NF-kappaB/Rel activation. This marker was created by targeted introduction of a lacZ reporter gene into the I-kappa-B-alpha locus. The second approach uses retroviral vectors tagged with a green fluorescent protein to reconstitute primary B-cells with specific forms of NF-kappaB/Rel proteins. Retroviral reconstitution will be used to determine how individual complexes regulate class switching and immunoglobulin secretion in B-cells. Defining the natural physiological roles of these factors will directly contribute to understanding why certain members are proto-oncogenes, and how their disregulation can lead to oncogenic activation in human cancer. Determining how these factors regulate immune responses will also produce a better understanding of the in vivo action of clinically relevant drugs, such as aspirin and glucocorticoids, that have been identified as inhibitors of this transcription factor family. Specific Aim 1 will determine when and where NF-kappaB/Rel complexes are activated in vivo during the natural history of an immune response to a T-cell dependent antigen. Specific Aim 2 will examine in vivo how the absence of individual NF-kappaB/Rel factors disrupts effective immune responses and determine the cellular basis for defective responses. Aim 3 will define roles of specific NF-kappaB/Rel complexes in primary B-cells in regulating immunoglobulin secretion and class switching.

DESCRIPTION (provided by the applicant): Toxoplasma gondii is a widespread intracellular human pathogen in the same phylum Apicomplexa that includes the human pathogens Plasmodium (the cause of malaria) and Cryptosporidium. Although Toxoplasma is emerging as a model system for the study of intracellular parasitism; our understanding of how host resistance genes control Toxoplasma growth has lagged behind our understanding of how Toxoplasma genes regulate pathogen growth within host cells. A clear understanding of the mechanisms responsible for intracellular host defenses to Toxoplasma will provide useful insights into how this important class of human pathogens can be controlled in infected patients. A major block impeding the functional analysis of how host-cell gene expression controls intracellular Toxoplasma growth is the tack of sensitivity of in vitro assays that measure intracellular parasite growth. To address this issue, a novel FACS-based assay has been developed to measure growth of GFP-tagged Toxoplasma in host cells. This FACS-based assay is significantly more sensitive than previous Toxoplasma growth assays, and can measure the inhibition of parasite growth mediated by individual retroviral-expressed genes in un-stimulated host cells. This new assay permits examination of host-cell responses to Toxoplasma in greater detail than previously possible, and will be used in Aim 1 to determine the mechanism of action of known host resistance genes, particularly the IGTP family of interferon-induced genes whose mechanism of action is unknown, despite having been implicated in the pathogenesis of multiple intracellular pathogens. In proof of principle studies, we have also established that known host resistance genes can be identified in functional screens designed to identify novel host resistance genes. Thus, in Aim 2, functional genetic screens will be conducted using retroviral cDNA libraries to identify host resistance genes that control Toxoplasma infection by (1) active resistance of growth of the virulent tachyzoite form of Toxoplasma, and by (2) passive inter-conversion of the virulent tachyzoite form to the dormant bradyzoite form of Toxoplasma.