The undue susceptibility of human neonates to infections with intracellular pathogene may result in part from an intrinsic and selective immaturity in production of IFN-gamma by neo-natal T cells and in part from the failure of neonatal M0 to respond to IFN-gamma with increased TNF production. This proposal addresses the central hypothesis that priming of T cells, which occurs by previous antigen or mitogen- induced activation, and of M0, which occurs by exposure to IFN-gamma, acts in part by modifying cellular signal transduction mechanisms.
Aim 1 seeks to determine if the basis for diminished IFN-gamma production, in spite of normal interleukin 2 (IL2) production, by neonatal t cells: 1. reflects in large part the absence of previously primed T cells with """"""""memory"""""""" phenotype; 2. greater production of IFN-gamma by """"""""memory"""""""" T cells reflects more efficient mechanisms transducing the signal to transcribe IFN-gamma mRNA; or 3. alternatively reflects differences in nucleoprotein/chromatin configuration necessary for IFN-gamma transcription. T cells of """"""""memory"""""""" and """"""""non-memory"""""""" phenotype will be identified and purified using monoclonal antibodies; we will also determine if """"""""memory"""""""" T cells can be derived from """"""""non-memory"""""""" T cells by stimulation and propagation in vitro. Using these cells, T cell activation induced protein phosphorylation will be studied using 2D gels and phosphotyrosine antibodies to determine if phosphorylation occurs on tyrosine or serine/threonine; results will be correlated with IFN gamma production and differences in lymphocyte-specific tyrosine kinase (lck) and protein kinase C (PKC) subtype expression and activity using specific cDNA probes, antibodies and functional assay. Alternatively, if correlative differences in signal mechanisms are not confirmed, IFN-gamma compared to IL-2 gene nucleoprotein/chromatin structure will be assessed by DNase mapping and gel retardation assays.
Aim 2 seeks to explore, in a manner analogous to the T cell studies, the role of enhanced/altered signal transduction by PKC and the myeloid specific tyrosine kinases hck and fgr in IFN-gamma enhancement of M0 TNF production. Activation induced protein phosphorylation and expression and activity of these kinases will be correlated with IFN-gamma enhancement of TNF production. If an association between increased expression/activity of a specific kinase and IFN-gamma enhancement of TNF production is found, we will attempt to mimic these effects by introducing into M0 cell lines expression vectors containing that kinase(s). The proposed studies will dissect the lines expression vectors containing that kinase(s). The proposed studies will dissect the mechanisms by which IFN-gamma production by T cells and TNF production by M0 are regulated and enhance out understanding of the selective immaturity of these neonatal defenses.
Showing the most recent 10 out of 51 publications
