John A. Thompson - US grants

Interleukin 2 (IL 2), also known as T cell growth factor, is a lymphokine produced by amplifier T lymphocytes. The specific stimulation of immune T cells with antigen "activates" T cells to express IL 2 receptors. IL 2 binds avidly to its receptor and induces T cell replication. In vitro, IL 2 induces the proliferation of "activated" T cells capable of mediating a variety of differentiated effector functions. Similarly, we have recently shown that the administration of purified IL 2 in vivo can induce the growth of antigen-activated T cells, and thereby augment specific T cell function. Thus, by virtue of its potent action on lymphoid cells bearing IL 2 receptors, IL 2 may have significant therapeutic potential as a pharmacologic agent to augment specific T cell immunity in vivo. The specific aims of the laboratory project are: to determine the pharmacologic potential of exogenous IL 2 by defining the influence of dose, route, and timing in inducing the growth and augmenting the function of specifically immune donor T cells in vivo; and to determine the effect of the IL 2 regimens developed on immune responses in both normal and immunodeficient mice.

A wide variety of projects in biomedical research require separating compounds by high performance liquid chromatography (LC) and subjecting these isolated species to structure analysis by mass spectrometry (MS). The development of thermospray techniques have greatly facilitated the interfacing of these instruments, so that online mass spectral analysis of chromatographic effluents can now be accomplished routinely. At the present time, however, no LC/MS system is available to the scientific community in the State of Colorado. Recognizing the potential of this technique for enhancing their research, a group of 10 PHS-supported investigators from the University of Colorado (Boulder), the University of Colorado Health Sciences Center (Denver), the AMC Cancer Research Center (Denver) and Colorado State University (Fort Collins), have described their plans for utilizing LC/MS if a shared instrument was purchased as a result of this application. A secondary user group of 4 investigators (supported be 4 PHS grants) has been identified also. Specific applications of LC/MS to the funded research of the user groups include analyses of (1) reactive and potentially toxic species (catechols, quinones, peroxides and unsaturated aldehydes) produced during the metabolism of aromatic compounds and during lipid peroxidation, (2) glutathione conjugates of reactive metabolites, (3) DNA adducts formed by interactions with toxic chemicals and anti-cancer drugs, (4) anthracyclines, (5) fragments of peptide neurotransmitters, (6) oligosaccharides, (7) glycolipids and (8) phospholipids. The strength of the LC/MS technique is in the analysis of compounds such as these that are nonvolatile, unstable or present in very low concentrations. The user group proposes to install an LC/MS system in the School of Pharmacy on the campus of the University of Colorado, Boulder. This is the most convenient location for the majority of users, and will be maintained by a group that has successfully maintained a gas chromatograph-mass spectrometer for several years. An internal advisory committee has been organized to assist the principal investigator, and a viable plan has been proposed to assure the long-term operation of a shared instrument.

DESCRIPTION (Adapted from Investigator's Abstract): Prominent angiopathologic events associated with hypertrophy and ischemia are accompanied by secretion of growth factors from reactive cells. The maintenance of endothelial integrity includes the responsibility of acidic fibroblast growth factor (FGF-1), an angiogenic polypeptide lacking a classical signal sequence for secretion. Recent evidence from this laboratory has defined a nonconventional pathway for cellular secretion of FGF-1 that is induced by oxidative stress associated with inflammation and mediated by superoxide, nitric oxide, and their reaction product, peroxynitrite. During this process, FGF-1 modulates the induction of oxidant-mediated apoptosis, an observation accompanied by the appearance of select polypeptides containing nitrated tyrosine residues. Collectively, these observations imply a central role for FGF-1 during resolution of inflammation and repair. Experimental aims are designed within the framework of the hypothesis that in response to oxidative stress, FGF-1 mediates its transforming potential through an extracellular pathway which can be attenuated with specific antioxidant agents. An underlying theme includes quantitating the production and targeted molecular responses to reactive nitrogen/oxygen species in vitro that modulate antioxidant defense mechanisms and FGF-1 signal transduction pathways. The availability of antibodies, nucleic acid probes/amplimers, recombinant proteins, expression vectors, and established techniques permits fundamental mechanistic studies of responding molecular cascades by analyses of mRNA (RT-PCR, hybridization) and protein (Western, ELISA, immunohistochemistry, enzymatic activity) expression. The opportunity to dissect and modulate key FGF-1 and oxidant-induced responses in defined vascular cells is complemented by the ability to extend these observations into relevant rodent models of hypertrophy and ischemia reperfusion injury. Elucidation of the molecular events responsible for the cause and effect relationship between oxidant stress and FGF-1 secretion and biologic activity will reveal detailed characteristics of these interrelationships, provide diagnostic criteria for monitoring this process, and establish more rational strategies of therapeutic intervention.

Advances in immunosuppressive therapy for transplantation have done little to decrease the percentage of allografts which eventually develop chronic rejection (CR). Studies proposed in this application collectively serve as a preclinical bridge with direct relevance to organ transplantation. The experiments are designed within the framework of the hypothesis that in response to oxidative stress associated with superoxide nitric oxide, and their reaction product, peroxynitrite, acidic fibroblast growth factor (FGF-1) mediates a transforming potential during chronic renal allograft rejection by an extracellular pathway. An underlying theme of these studies includes quantitating the production and targeted molecular reactions of reactive nitrogen/oxygen species that inactivate antioxidant defense mechanisms (manganese superoxide dismutase and glutathione) in the kidney allograft to compromise renal structure/function. To establish: (a) a cause-and-effect relationship between oxidant stress, FGF-1, and CR: (b) a diagnostic criteria for monitoring this process; and (c) therapeutic interventional strategies, the administration of neutralizing antibodies against FGF-1 and antioxidants will be evaluated for their ability to modulate the kinetics of developing CR lesions in an established allogeneic rat model of kidney transplantation. The availability of antibodies, nucleic acid probel/amplimers, and established techniques permits the dissection of responding molecular cascades during CR by analyses of mRNA (RT-PCR, hybridization) and protein (Western, ELISA, immunohistochemistry, enzymatic activity) expression in the kidney. Special emphasis will focus on the ability of FGF-1 to modulate growth (PCNA), peroxynitrite-induced apoptosis (TUNEL), and tyrosine nitration in target proteins. The correlation of urinary appearance of FGF-1, nitrite/nitrate, albumin, and nitrotyrosyl proteins with in situ development of pathologic lesions will form the basis of a non-invasive diagnostic assay for monitoring CR during renal transplant dysfunction. While the precise involvement of FGF-1, reactive nitrogen/oxygen species, antioxidants, and nitrotyrosyl proteins are not known, efforts within this application will elucidate detailed characteristics reflective of pathophysiologic processes associated with CR not only in the rat model but also in kidney transplanted primates and human patients undergoing immunosuppressive therapies.

DESCRIPTION (Provided by applicant): Current treatments for metastatic melanoma are unsatisfactory, as very few patients achieve durable complete remissions. This project explores a novel concept for delivering immunotherapy, via nonmyeloablative allogeneic stem cell transplantation (Allo SCT). We hypothesize that the establishment of donor hematopoietic chimerism will induce a graft-versus-tumor (GVT) effect. A growing list of antigens have been identified on melanoma cells, which may elicit Class I or Class II T cell responses. The mechanism of GVT effect after Allo SCT is under intense scrutiny, and may involve T cell reactivity to minor histocompatibility antigens, as well as to tumor specific antigens. The Specific Aims of this proposal are: 1. Complete the first stage of a Phase II trial (FHCRC protocol 1462) of Allo SCT in patients with metastatic melanoma, to determine the clinical efficacy (as assessed by the objective response rate and survival) and toxicity (including graft-versus-host disease). Up to 15 patients with metastatic melanoma, who have achieved partial remission, minor remission, or stable disease after initial systemic therapy, will be treated. Eligible patients will receive low-dose total body irradiation (200cGy), and fludarabine, followed by infusion of peripheral blood stem cells from a HLA matched sibling donor, with post-transplant cyclosporine and mycophenylate mofetil. Subsequently, selected patients will receive donor lymphocytes to augment the GVT effect. 2. Evaluate immune responses, in patients undergoing Allo SCT, to known melanoma antigens, novel tumor-specific antigens, tissue-specific antigens, and minor histocompatibility antigens. The CD4+ and CD8+ T cell responses to defined peptides will be assessed by tetramer analysis, cytolytic effector function, and proliferative reactivity. Evaluation of the potential response, safety of Allo SCT, and elucidation of these T-cell responses will facilitate the development of future studies to manipulate GVT effect in patients with metastatic melanoma.

DESCRIPTION (provided by applicant): Application of steroid-free immunosuppressive protocols has markedly improved outcomes in pancreatic isle transplantation (PIT). However, large numbers of islets are required to achieve insulin-independence. While approximately 1 million islets exist in the adult human pancreas, the current pancreas preservation and islet isolation techniques recover only 20-50% of the islets. In addition, a significant reduction of islet mass occurs in the peritransplant period. Presently, cadaver donors remain the sole source of pancreatic tissue for PIT. Unfortunately no studies have been conducted to date to determine the biological effects of BD on isolated human islets. Moreover by recommendation of the American Society of Transplant Surgeons, multiorgan donors receive triiodothyronine vasopressin, cortisol and insulin. However, no information is available regarding beneficial or detrimental effects oft human islets. Our recent studies indicate that brain-death (BD) is associated with decreased isolated islet recovery functionality, and engraftment after transplantation. Deleterious effects of BD on isolated islets could explain the major differences observed in clinical PIT outcomes compared with results routinely obtained in experimental animals. Based on the anti-inflammatory, anti-apopototic, and anti-oxidant properties of 17beta-estradiol, we previously demonstrated significant reduction of proinflammatory cytokine-mediated human islet death. In addition, our preliminary data demonstrate that estradiol administration to BD-donors increases islet recovery in rats. It is conceivable to postulate that reduction of deleterious effects of BD by 17beta-estradiol treatment to human BD donor; would mitigate islet loss, improve islet functionality and engraftment after transplantation. Our studies will examine the hypothesis that BD is associated with decreased human islet yields, viability, functionality and engraftment. To this end, islets obtained from BD-donors will be compared with islets recovered from human patients subjected to pancreatectomy for benign pancreatic tumors. In addition, we will analyze islet preparations obtained from BD-donors treated with either vehicle, estradiol or "hormonal therapy". Extensive molecular studies including islet gene expression profiles using microarrays, islet protein kinases using multi-immunoblotting techniques will provide information about the molecular mechanisms involved in detrimental effects of BD and will identify potential therapeutic targets. Overall, these studies will bring novel information about human islet biology and will provide c potential therapeutic strategy to enhance the therapeutic efficacy of PIT as a cure for diabetes.

Project Summary Parkinson?s disease (PD) is a neurodegenerative disorder that leads to both motor and non-motor symptoms. While there is as yet no cure for PD, medical and surgical therapies have been developed that effectively target the motor symptoms of PD. Non-motor symptoms are far more disabling for patients, precede the onset of motor symptoms by a decade, are more insidious in onset, have been less apparent to clinicians, and are less effectively treated. Sleep dysfunction is oftentimes the most burdensome of the non-motor symptoms?both to patients and to their caregivers?is pervasive in patients with PD, and includes sleep fragmentation, insomnia, excessive daytime sleepiness, REM behavioral disorder, and restless leg syndrome. There are limited options for treating sleep dysfunction in PD, and the mainstay of therapy is the use of agents that mask the sleep disturbance?such as the sedative-hypnotic drugs?without addressing the underlying mechanisms. Although much attention has been devoted to PD motor symptoms, sleep dysfunction in PD has largely been ignored. Sleep is vital to homeostasis, cognition, and nervous system repair, and the dysfunctional sleep accompanying PD adversely affects both motor and non-motor symptoms, resulting in diminished quality of life, impairments in mood and behavior, and increased morbidity and mortality. Patients with PD who demonstrate significant motor fluctuations and dyskinesia are considered for subthalamic nucleus (STN) deep brain stimulation (DBS) surgery. Although STN-DBS is routinely used to treat PD motor symptoms, several studies have reported that STN-DBS also provides benefit for sleep dysregulation through normalization of sleep architecture. Additionally, local field potentials recorded from STN DBS electrodes implanted for the treatment of PD, have led to the identification of unique spectral patterns in STN oscillatory activity that correlate with distinct sleep cycles, offering insight into sleep dysregulation. Building on this work, and in response to RFA-NS- 18-023, this proposal will leverage novel investigational DBS battery technology (RC+S Summit System; Medtronic) that allows the exploration of sleep biomarkers and prototyping of closed-loop stimulation algorithms, to test the hypothesis that STN?a highly interconnected node within the basal ganglia?contributes to the regulation and disruption of human sleep behavior and can be manipulated for therapeutic advantage. Specifically, in PD patients undergoing STN-DBS, we will determine whether STN oscillations correlate with sleep stage transitions, then construct and evaluate sensing and adaptive stimulation paradigms that allow ongoing sleep-stage identification, and induce through adaptive stimulation an increase in duration of sleep stages associated with restorative sleep. This work will lead to findings that address a currently unmet clinical need, and relevant to the mission of NINDS and the BRAIN Initiative, will evaluate the use of adaptive stimulation of the STN in PD patients for the treatment of sleep dysfunction.