Stephen Miller - US grants

With this award co-funded by the Division of Chemistry and the Major Research Instrumentation (MRI) program, Professor Daniel R. Talham and colleagues John R. Reynolds, K. B. Wagener and Stephen A. Miller from the University of Florida and Kevin D. Belfield of the University of Central Florida will acquire a matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometer (MALDI TOF-TOF MS). The award will enhance research, training and education at all levels, especially in polymer science, chemical imaging, biomarker discovery and natural products research. Specific areas of research to be investigated include studies of acyclic diene metathesis of precision olefins, multiphoton absorbing supramolecular photonic materials, biodegradable plastics, conjugated donor-acceptor polymers, peptide-based natural products, and bioanalytical applications of aptamer-based platforms.

Mass spectrometers (MS) are used to identify the chemical composition of a sample by measuring the mass of the molecular constituents in the sample after they are ionized and detected by the mass spectrometer. Matrix-assisted laser desorption ionization (MALDI) is a technique for preparing the sample to be ionized by a laser before injection into the mass spectrometer. The time of flight (TOF) mass analyzer has high sensitivity and mass accuracy to assist the analyses and interpretation of the resultant data. This open access instrument will be available to all at the Universities of Florida and Central Florida and as a service for external user groups throughout the Southeast and Puerto Rico, including several at Florida A&M University. It will provide training to undergraduate, graduate and post doctoral students in state-of-the-art mass spectrometry usage.

? DESCRIPTION: The undesired destruction of healthy cells by the immune system results in the loss of tissue function and complicates strategies to restore tissue function. The current standard therapy for autoimmune disease involves generalized immunosuppression, which is in most cases is not clinically efficacious and leads to numerous undesired side effects. Dr. Stephen Miller, (co-PI) pioneered an approach in which splenocytes were cross-linked with specific auto antigens, and their delivery to the spleen induced tolerance specifically to the auto antigen. This approach was recently adapted for a clinical trial in multiple sclerosis (MS) patients, and was the first-in-man study to report the induction antigen specific tolerance. However, the use of cellular carriers for tolerance induction in the clinical arena is challenging due to the considerable ex-vivo laboratory manipulation that is required, which is expensive, increases the number of donor cells needed and introduces further opportunity for technical error. Our long-term goal is to develop a particle-based platform that can be an off-the-shelf product for induction of tolerance to specific antigens to inhibit the specific undesired immune response while not altering the remaining elements of the immune response. We have demonstrated that antigen-loaded particles delivered intravenously can induce tolerance for the prevention and treatment of experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. With the goal of moving this technology toward the clinic, we propose to extend these studies to address fundamental questions about the particle design and their mechanisms of action, and also critical questions regarding the ability to target the variety of antigens and cell populations underlying disease. Specific Aim 1 will investigate the particle design parameters and identify the cellular mechanisms by which particles injected intravenously are able to modulate inflammation and induce antigen specific tolerance. Our results suggest the liver as a critical site involved in tolerance induction from the particles, which distinguishes it from the previous work with antigen-coupled splenocytes. We propose to investigate the particle composition and size to distinguish i) the impact of the carrier on immune cell polarization, ii) te efficacy of antigen presentation, and iii) in vivo trafficking of the particles. Specific Aim 2 wil determine the cellular and molecular mechanisms by which Ag-PLG tolerance is induced and maintained in naïve, activated, and memory T cells. We propose to test the ability to induce tolerance with particles encapsulating multiple peptides/proteins and to examine the separate and combined contributions of anergy and Tregs to the induction and maintenance of tolerance. Successful completion of these studies would identify particles that are novel, safe, efficient and clinically relevant tools to inhibit antigen-specific T-cells for therapy of autoimmune diseases. This innovative approach has far reaching implications for decreasing specific immune responses in applications such as autoimmune disease, rejection of transplanted cells, and allergies to food antigens or airborne particulates.

DESCRIPTION (provided by applicant): The long-term goal of the proposed preclinical studies is to develop a clinically applicable tolerogenic protocol for use in human islet allotransplantatin in T1D. The central component of our strategy is the delivery of antigens on leukocytes treated with the chemical cross linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). Autoantigen-coupled splenocytes given IV prevent and treat autoimmunity in mice. In transplant models, ECDI-fixed donor splenocytes given IV on days -7 and +1 - as induce long-term donor-specific tolerance to islet allografts, and when combined with short-term rapamycin (RAPA), also to heart allografts in mice. A first-in-human clinical trial of autologous, peptide-coupled cels in multiple sclerosis (MS) recently established the clinical feasibility of this novel tolerogenic strategy. To test whether the profound tolerogenic efficacy of alloantigen delivery via ECDI-fixed cells (ADEC) will translate to islet transplantation in nonhuman primates (NHP), we will study the following specific aims: AIM #1: To manufacture ADEC products meeting prospectively defined release criteria for evaluation as tolerogens in islet allotransplantation in RM. AIM #2: T determine the efficacy of ADEC in inducing tolerance to islet allografts in RM with low and high memory alloreactivity transiently treated with RAPA, sTNFR, ¿-IL-6R, and LFA3-Ig. AIM #3: To examine the effects of the immunotherapeutic protocol on mechanisms underlying the induction, maintenance, and/or loss of donor-specific tolerance to islet allografts in RM. The innovation of this proposal lies expressly in the preemptive use of potent, yet safe, cellular immunotherapeutics as antigen-specific, negative vaccines. Our protocol targets innate, heterologous, and adaptive direct and indirect pathway immunity (and can be extended to target autoimmunity) and has, despite complete avoidance of generalized T and/or B cell depletion and costimulation blockade, a high potential for inducing durable tolerance to islet allografts in NHP. The proposed studies will provide novel insights into the role of ADEC and concomitant immunotherapy for tolerance induction to islet allografts, a critical step toward clinical translaton of this antigen-specific tolerance strategy. PUBLIC HEALTH RELEVANCE: Human islet allotransplantation restores insulin independence and near normoglycemia, protects from severe hypoglycemia and slows the progression of microvascular complications in those people with type 1 diabetes (T1D). The numerous undesirable side effects and the high costs of chronic immunosuppression limit the applicability of islet transplants. To overcome this limitation, the goal of this proposal is to develop in a pre clinical model of T1D (diabetic non-human primates) tolerogenic protocol(s) for use in human islet allotransplantation in T1D.