We hypothesize that β-cell toxicity in T1D can be induced via different pathways by distinct pathological ligands (AGEs, S100-proteins, amyloidogenic amylin) of the receptor for advanced glycation endproducts (RAGE) that become upregulated in the diabetic pancreas. We also hypothesize that C-peptide (+) T1D subjects may be more susceptible to amylin-induced β-cell toxicity. Amylin is co-produced /co-secreted with insulin by β-cells; subjects that retain the ability to produce/secrete insulin may also produce/secrete amylin. We will test out hypotheses in three successive stages: 1) Method validation, 2) Hypothesis-driven proof-of-concept testing, and 3) Mechanistic studies guided by findings in Stage-2. Having completed Stage-1 using nPOD control pancreas sections (T2D and nondiabetic), we are ready to test our hypothesis in 4 phenotypes of human subjects (C-peptide(+) T1D, C-peptide(-) T1D, T2D and non-diabetic controls) according to two synergistic specific aims. Aim-1: We will determine whether RAGE lignads (AGEs, S100-proteins, amyloidogenic amylin) become upregulated in T1D islets. Aim-2: We will determine whether islet amyloidosis correlates with C-peptide levels in T1D. IHC-IF will be used to quantify RAGE ligands/signaling molecules (amylin/amyloid, AGEs, RAGE, mDia1) and loss in β-cell/islet area and apoptosis (insulin/synaptophysin, cleaved capase3) in pancreas sections. RAGE ligands and signaling molecules (S100A12, RAGE, mDia1) will also be measured in frozen pancreas specimens by qRT-PCR, and in plasma (S100B) by ELISA. Upregulated ligands/mediators identified in Stage-2 will be the focus of mechanistic studies in Stage-3; which aim to identify mediators of oxidative stress (NOX1, NOX4, NOS), inflammation (CCL2, TNFα, ILβ, IL6, IL10) and apoptosis (bax) in frozen pancreas and spleen (control) specimens viz qRT-PCR. Results will be biostatically compared/contrasted/correlated. Successful completion of the proposed studies will identify key pathological factors and associated mechanisms by which RAGE contributes to β-cell/islet pathology in T1D, providing important insight into new drug targets for β-cell preservation and treatment/prevention of this disease.