Neelima Shah

Basal Igα/Igβ Signals Trigger the Coordinated Initiation of Pre-B Cell Antigen Receptor-Dependent Processes

The pro-B to pre-B transition during B cell development is dependent upon surface expression of a signaling competent pre-B cell Ag receptor (pre-BCR). Although the mature form of the BCR requires ligand-induced aggregation to trigger responses, the requirement for ligand-induced pre-BCR aggregation in promoting B cell development remains a matter of significant debate. In this study, we used transmission electron microscopy on murine primary pro-B cells and pre-B cells to analyze the aggregation state of the pre-BCR. Although aggregation can be induced and visualized following cross-linking by Abs to the pre-BCR complex, our analyses indicate that the pre-BCR is expressed on the surface of resting cells primarily in a nonaggregated state. To evaluate the degree to which basal signals mediated through nonaggregated pre-BCR complexes can promote pre-BCR-dependent processes, we used a surrogate pre-BCR consisting of the cytoplasmic regions of Igα/Igβ that is targeted to the inner leaflet of the plasma membrane of primary pro-B cells. We observed enhanced proliferation in the presence of low IL-7, suppression of VH(D)JH recombination, and induced κ light (L) chain recombination and cytoplasmic κ L chain protein expression. Interestingly, Igα/Igβ-mediated allelic exclusion was restricted to the B cell lineage as we observed normal TCRαβ expression on CD8-expressing splenocytes. This study directly demonstrates that basal signaling initiated through Igα/Igβ-containing complexes facilitates the coordinated control of differentiation events that are associated with the pre-BCR-dependent transition through the pro-B to pre-B checkpoint. Furthermore, these results argue that pre-BCR aggregation is not a requirement for pre-BCR function.

Insulin-induced egr-1 Expression in Chinese Hamster Ovary Cells Is Insulin Receptor and Insulin Receptor Substrate-1 Phosphorylation-independent EVIDENCE OF AN ALTERNATIVE SIGNAL TRANSDUCTION PATHWAY

Insulins effects primarily are initiated by insulin binding to its plasma membrane receptor and the sequential tyrosine phosphorylation of the insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1). However, studies suggest some insulin effects, including those at the nucleus, may not be regulated by this pathway. The present study compared the levels of insulin binding, insulin receptor and IRS-1 tyrosine phosphorylation, and phosphatidylinositol 3′-kinase activity to immediate early gene c-fos and egr-1 mRNA expression in Chinese hamster ovary (CHO) cells expressing only neomycin-resistant plasmid (CHONEO), overexpressing wild type human insulin receptor (CHOHIRc) or ATP binding site-mutated insulin receptors (CHOA1018K). Insulin binding in CHONEO cells was markedly lower than that in other cell types. 10 nM insulin significantly increased tyrosine phosphorylation of insulin receptor and IRS-1 in CHOHIRc cells. Phosphorylation of insulin receptor and IRS-1 in CHONEO and CHOA1018K cells was not detected in the presence or absence of insulin. Similarly, insulin increased phosphatidylinositol 3-kinase activity only in CHOHIRc cells. As determined by Northern blot, nuclear run-on analysis, and in situ hybridization, insulin induced c-fos mRNA expression, through transcription, in CHOHIRc cells but not in CHONEO and CHOA1018K cells, consistent with previous reports. In contrast, all three cell types showed a similar insulin dose-dependent increase of egr-1 mRNA expression through transcription. These data indicated that insulin-induced egr-1 mRNA expression did not correlate with the levels of insulin binding to insulin receptor or phosphorylation of insulin receptor and IRS-1. These results suggest that different mechanisms are involved in induction of c-fos and egr-1 mRNA expression by insulin, the former by the more classic insulin receptor tyrosine kinase pathway and the latter by a yet to be determined alternative signal transduction pathway.

779. Oral Delivery of Non-Viral DNA Nanoparticles for Hemophilia A

Hemophilia A is a defect in coagulation factor VIII, which is predominantly produced in liver and secreted into plasma at a level of 100-200 ng/ml. Hemophilia A is an attractive model for non-viral gene delivery since levels as low as 1-2% FVIII can affect bleeding. In addition, FVIII can be processed from non-liver cell types. While non-viral gene delivery has advantages of production and safety compared to viral gene therapy, the transient nature of gene expression makes re-administration necessary, therefore an oral route of administration highly desirable. We previously reported preliminary results in hemophilia A mice orally administered chitosan-FVIII DNA nanoparticles, including phenotypic bleeding correction in 4/5 mice given the highest DNA dose. Levels of functional FVIII by chromogenic assay were modest. However a thrombin assay revealed significantly greater thrombin generation in mice given chitosan-DNA nanoparticles as compared to naked DNA.