The predicted increase in the number of people worldwide afflicted with T1 and T2 diabetes (approximately 230 million during the next 20 years) necessitates the ongoing effort to develop improved methods for the early diagnosis of the disease, which may afford greater success with early clinical intervention and ultimately, disease prevention. It is increasingly evident that therapeutic interventions at disease onset have the greatest likelihood of reversing the otherwise continuously progressing destruction of insulin-secreting -cells by the immune system. It is now known that both Type 1 (the early insulitis stage) and type 2 diabetes share an initial pathology that starts with microenvironmental changes brought on by pro-inflammatory signaling and associated local vascular dysfunction; -cells death, which occurs during the initiation and progression of both type 1 and type 2 diabetes, is mediated by the inflammatory signaling molecules IL-1 and TNF?, which in turn are produced subsequent to the activation of NF-?B, a transcription factor central to many inflammatory signaling pathways. In view of the above we are confident that the ability to non-invasively detect early changes in functional NF-?B eventually will provide a means to predict patients who may be in the earliest stage of potentially irreversible damage to endocrine pancreas. In addition, functional analysis of activated NF-?B will provide a sensitive means for studying the longitudinal effects of newly developed therapeutics designed to slow or prevent the development of this disease. Our recent work has validated the use of stabilized near-infrared (NI) fluorescent oligonucleotide duplex- based molecular sensors (ODN-MS) created using new approaches in bioconjugate chemistry as sensors for detecting activated NF-?B in vitro. The detection is afforded by changes in NIR photon emission profiles evoked by NF-?B-DNA binding events occurring at the genomic level. In a parallel line of investigation, we have designed, synthesized, and tested nano-sized protected graft co-polymer (PGC) as a carrier for the delivery of paramagnetic and optical probes to the vascular compartment of pancreas in a mouse model of diabetes (Medarova Z, et al. Diabetes 2007; 53:1318-25). Here we propose, in collaboration with investigators from the MGH A. Martinos' Center, to harness the combined strength of both novel probe technologies to design synthesis and test a hydrophobic core (HC) PGC-ODN molecular sensor capable of detecting molecular interactions between the transcription factor NF-?B and DNA. We will further test the applicability of the probe for clinica translation by determining its delivery and cellular uptake in the pancreas in a murine model of diabetes. And finally we will test the sensitivity of the PGC-ODN probe for detecting changes in NF-?B-DNA binding events by performing diagnostic fluorescence lifetime in vivo imaging of NF-?B activity in the pancreas. Toward achieving this goal we propose to pursue the following aims:
Aim 1 : use in silico modeling and a novel combination of interbase linker and end-linker approaches for designing optimized ODN-MS probes with improved cell delivery;
Aim 2 : test delivery of ODN-MS in vitro performing cell and isolated islet-based assays and biodistribution studies in an acutely diabetic mouse model;
Aim 3 : investigate in vivo fluorescence lifetime imaging as a modality for determining the extent of NF-?B activation in normal and diabetic mice using a state of the art imaging platform developed at the MGH A. Martinos' Center. The sensitivity of the imaging method to detect disease burden will assessed by controlling the extent of disease via a therapeutic agent (an I?B phosphorylation inhibitor of TNF? signaling, BAY-11- 7082), which alters levels of NF-?B expression and prevents the destruction of islet cells in NOD and NOD/Lepr mice.
It is estimated that if the current trend will not change, there will be about 230 million cases of diabetes worldwide during the next 20 years. Even assuming successful preventive efforts in fighting juvenile obesity in the future and drastic changes of the diet, the increase in a number of cases of diabetes has global implications and will require major changes in the ways we diagnose and treat this disease. Understanding of pathogenesis of diabetes is critical in finding the cure. We propose to research special imaging sensors that are capable of detecting early changes in pancreatic cells that secrete insulin and other essential factors regulating glucose metabolism. These sensors are needed in very small amounts for telling whether the immune system started attacking pancreatic cells that leads to diabetes. The sensors are activated by light and do not emit or require exposure to ionizing radiation. This potentially will enable interfering with the progression of the disease at very early stages ultimately helping in saving lives of patients that otherwise would progress to the full-blown disease.
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