Type 1 diabetes mellitus (T1DM) is a T cell-mediated autoimmune disease, and the most prevalent pediatric autoimmune disorder. Its primary pathology is the complete immune cell-mediated destruction of the insulin-producing pancreatic beta cells. This process occurs upon the infiltration of the pancreatic islets by leukocytes, in a process termed insulitis, which selective depletes insulin-producing beta cells. When this deficient becomes critical, frank diabetes is observed as the acute onset of hyperglycemia. The immunopathology of T1DM is well modeled in the non- obese diabetic (NOD) mouse, which has emerged as the """"""""gold-standard"""""""" of autoimmune diabetes research. What is most vexing in both the human clinical setting and the NOD research model, is the lack of a clear indicator of sub-clinical insulitis and its severity, as the insulitis process is occult and occurs over a prolonged period of time in the total absence of overt symptoms. The inability to time and stage insulitis has greatly hampered research into T1DM etiology and molecular pathogenesis. Therefore there is a pressing need for a vital and real-time measure for insulitis. To address this, our goal is to product an NOD mouse that signals the initiation and severity of insulitis by means of a molecular report that is readily, reliably and rapidly detectable by a minimally-invasive and non-lethal means. This reporter must allow for a rapid and cost-effective qualitative assessment of insulitis. Soluble human placental alkaline phosphatase (sHPAP) is an ideal candidate. It has excellent bio-availability and stability in the blood, it has a unique substrate, an existing and sensitive fluorimetric assay for quantitative detection, and it is resistant to inhibitors of mouse alkaline phosphatase. Our previous work, using functional genomics, has identified Reg3gamma as a gene that undergoes rapid and sustained up-regulation upon the initiation of insulitis in the NOD mouse. This beta cell-specific gene has a protective, anti-apoptotic role in beta cell survival and has no to very low basal expression in uninfiltrated islets but exhibits up to a 30-fold progressive increase in expression with insulitis. Here we proposed to make knock-in NOD mice that use the endogenous Reg3gamma promoter to drive the expression of sHPAP in pancreatic beta cells. As beta cells are specialized secretory cells with intimate access to the blood, we hypothesize that upon the natural immune-infiltration of islets, beta cells will produce sHPAP. We can then quantify sHPAP from a small blood sample of NOD mice to determine the initiation and severity of insulitis. To this end, we propose the following specific aims:
Specific Aim 1 : To product insulitis reporter (InsuRe) NOD mice by the targeted introduction of soluble human placenta! alkaline phosphatase into the Reg3gamma locus.
Specific Aim 2 : To validate the specificity and sensitivity of the insulitis reporter NOD mice by qualitative analysis of sHPAP expression and documented insulitis in NOD mice. ? ? ?
