The proposed studies seek to analyze an important subset of the transcriptome and proteome in mouse models of type I diabetes (T1D), specifically characterizing the transcriptome and proteome of pancreatic lymph node (PLN) cells, islet beta cells, and peripheral blood cells over time. We will then use a systems biology approach to perturb the system through treatment of mice with anti-CD3 monoclonal antibodies, which have been shown to reverse hyperglycemia in newly-diabetic mice, and are currently in clinical trials in human beings. We hypothesize that the transcriptome and proteome will revert to normal in treated mice, or will instead identify a """"""""diabetes proteomic biosignature"""""""" that will further our understanding of the mechanisms of action of anti-CD3. These studies will employ two unique and cutting-edge technology platforms (eTag capillary electrophoresis assays, and reverse phase protein mysate microarray (RPPLM) assays) for studying a subset of the transcriptome and proteome, respectively. We will draw on unique resources existing at Stanford University to complete these studies, including collaborations with Dr. C. Garrison Fathman (expertise in T1D and genomics), Dr. Garry Nolan (expertise in immunology, fluorescence activated cell sorting or FACS, and proteomics) and Dr. Robert Tibshirani (expertise in proteomics and biomedical informatics). This team will test the ability of a combined genomic and proteomic analysis to obtain a clear and reproducible signature pattern of progressive stages in the development of T1D. These assays will be applied to 4 related specific aims: (i.) to analyze multiple signaling pathways in purified CD4+ T cells from the PLNs of NOD.BDC2.5 mice and NOD.B10 mice, sacrificed at different time points reflecting the prediabetic and overtly diabetic state; (ii.) to determine the activation status of multiple signaling proteins in CD4+ T cells isolated from NOD mice treated with anti-CD3, and to compare the signaling proteome between untreated and treated mice; (iii.) to use laser capture microdissection (LCM) to analyze the signaling proteome of islet-infiltrating lymphocytes and beta cells; and, (iv.) to determine whether peripheral blood CD4+ T cells reflect similar changes in the transcriptome and proteome. If these patterns are similar, then these assays may provide a reliable surrogate T cell marker for disease progression and for assessment of therapeutic response in humans. This work is soundly within the scientific mission of the NIH, and is solely aimed at studying NOD mice, one of the most useful mouse models for T1D that exists. Our long term goal is to use the same approach for studying blood cells derived from human diabetic patients, hopefully allowing us to categorize patients and to define novel patient-specific or target-specific therapeutic modalities. ? ?
| Wu, Jenny C F; Chruscinski, Andrzej; De Jesus Perez, Vinicio A et al. (2009) Cholinergic modulation of angiogenesis: role of the 7 nicotinic acetylcholine receptor. J Cell Biochem 108:433-46 |
