Project 1: In previous studies we have defined immunologic abnormalities associated with the hyper-IgM syndrome, including those occurring in a rare X-linked form of the syndrome associated with hypohydrotic ectodermal dysplasia (XHH-ED). During this period we have defined the molecular defect in this latter disorder. In the relevant studies we focused on the structure and function of nuclear factor kappa B (NF-kappa B) essential nodular (NEMO or IKKgamma) an X-linked gene that could conceivably be important in signaling via CD40 and a related receptor, DL, which has been shown to be involved in ectodermal dysplasia in mice. Indeed, we found that patients with XHM-ED had mutations in the putative zinc-finger domain of NEMO that prevent CD40 ligand (CD40L)-mediated dysregulation of inhibition of NF-kappa B alpha (IkappaB-alpha). As a result, B cells from XHM-ED patients are unable to undergo immunoglobulin class-switch recombination to their antigen-presenting cells (APC's) are unable to synthesize NF-kappaB-regulated cytokine IL-12 to TNF-alpha when stimulated with CD40L. Interestingly, however, innate immunity is preserved in XHM-ED patients since APC's retain the capacity to respond to stimulation by lipopolysaccharide or Staphylococcus aureus Cowan's antigen (SAC). Overall, the phenotype observed in XHM-ED patients shows that the putative zinc-finger domain of NEMO has a regulatory function and demonstrates the requirement of CD40-mediated NF-kappaB activation for B cell immunoglobulin class-switching. In further studies we have used cDNA microarray technology to search for target genes of NF-kB signaling that modulates class switch recombination in B cells. In these studies we stimulated B cells from XHM-ED patients and normal controls with CD40L and IL-4 and compared the expression level of more than 12,000 genes. These experiments revealed approximately 50 genes were consistently not induced in patients with XHM-ED. The identity of these genes reveals that CD40L signaling in B cells may affect: cell growth, cell cycle, genetic recombination, and cell signaling. Our approach of utilizing comparative genomics technology to the study of this rare genetic disease, has allowed us to deconstruct a complex signaling pathway essential for B cell terminal differentiation, which would otherwise be difficult to reveal. We are now characterizing interesting genes not previously implicated in CSR and may be involved in recognizing and modifying the RNA/DNA template at the switch region. Project 2: As reported earlier, we have instituted a broad-based clinical study of the use of recombinant CD40L in patients with X-linked hyper IgM syndromes due to CD40L deficiency or dysfunction. We are presently in the midst of this study having treated approximately 5 patients with XHM with recombinant CD40L. We have found that the drug is well tolerated and have some evidence that it restores the ability of pateints' cells to produce IFN-gamma in vitro.
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