Stephen Tomlinson - US grants

Trypanosoma brucei brucei, unlike the human pathogens Tau beta rhodesiense and Tau gambiense, is lysed by human serum. Sensitivity to human serum is the only method which allows distinction between the cattle and human African trypanosomes. We have identified two distinct trypanolytic fractions in normal human serum (NHS) which differ physically, biochemically and with regard to their inhibition by serum factors. Significantly, an HDL-like trypanolytic factor (TLF1) described by others is not active in NHS, and only becomes lytic upon inhibitor removal during isolation procedures. It is proposed to characterize the more physiologically relevant lytic factor in human serum, and to determine the mechanism(s) by which parasites are killed by both factors. Data indicates that a mechanism of lysis may involve oxidative stress and subsequent programmed cell death, and these possibilities will be tested directly. This work may open new perspectives for the chemotherapy of trypanosomiasis. Understanding of the mechanisms of cytotoxicity and the affected metabolic pathways may provide new approaches for development of specific drugs.

DESCRIPTION: (Adapted from the Investigator's abstract): The broad long term objective of the proposed research is to develop novel complement inhibitory proteins for the effective and safe treatment of autoimmune and inflammatory disease. The specific aims of this application are to: 1. Prepare and characterize complement inhibitory proteins that can be targeted to specific tissues or sites of disease. Different classes of complement inhibitor will be fused to a targeting component consisting of either an antibody variable region or a receptor ligand. 2. Evaluate the therapeutic potential of targeted complement inhibitors in a murine model of spontaneous systemic lupus erythematosus (SLE). 3. Determine the effect of systemic complement inhibition on host ability to control an infection during chronic disease. Soluble CD59 or DAF functional units will be fused to either antibody fragments containing a variable region specific for a cell surface molecule, or to a soluble ligand recognizing selectins. Selectins are cell adhesion molecules that are expressed on inflamed endothelium and are involved in leukocyte recruitment. Various construct designs will be evaluated in vitro for selective targeting to a cell surface, and for their functional effectiveness at inhibiting complement and complement-dependent inflammatory processes. Complement inhibitory proteins based on antibody fusions that are effective in vitro will be tested in vivo in a murine model. A characterized murine anti-DNA antibody that has been shown to target to the kidney will be used for the preparation of IgG-complement inhibitor fusion proteins. Anti-DNA antibodies of the kind that will be used are relatively specific for lupus and have a pathogenic role in disease. Studies will determine the relative effects of targeted vs. untargeted (systemic) complement inhibitors, as well as the effect of selectively blocking the generation of different complement activation products. In addition to therapeutic evaluation, the proposed studies will allow testing of certain hypotheses relating to complement-associated disease mechanisms and the clinical use of complement inhibitors.

DESCRIPTION (provided by applicant): CD59 is a membrane-bound glycoprotein that inhibits the formation of the complement membrane attack complex (MAC or C5b-9) on host cells. CD59 functions by binding to C8alpha and C9 in the assembling MAC and interfering with its membrane insertion. Inappropriate complement activation and MAC deposition is involved in the pathogenesis of many autoimmune and inflammatory diseases, and is involved in hyperacute rejection of xenografts. Understanding the molecular interaction between CD59 and its ligands will provide a foundation for designing modified and improved complement inhibitors. In addition, CD59 expressed on tumor cells has been implicated in promoting tumorigenesis. The accomplishment of aims will enable design of small molecules to effectively block the function of CD59 on tumor cells. The goal of the application is to define the molecular interaction between CD59 and its C8 and C9 complement protein ligands. Specifically it is proposed to: 1. Identify the specific CD59 residues that are important for function. This aim will be accomplished using systematic scanning mutagenesis as well as rational substitutions of important regions and residues, with assistance from molecular modeling. 2.ldentify CD59 residues directly involved in ligand binding to determine whether C8 and C9 share the same binding residues on CD59, and to determine the nature of the species selective interaction between CD59 and its ligands. 3. Identify the C8 and C9 residues that bind to CD59 by analysis of effects of mutagenesis within identified C8/C9 binding regions. 4. Determine the three-dimensional structure of the CD59-C8 and CD59-C9 peptide ligand. Molecular modeling techniques together with experimental determinations by NMR will be utilized.

DESCRIPTION (provided by applicant): Complement is a crucial component of the immune system that can be involved in an effector response to tumor cells, and can also enhance the induction phase of a humoral immune response. Nevertheless, immunotherapy using complement activating antibodies that are specific for tumor-associated antigens has met with only limited success. A significant factor in the resistance of tumor cells to antibody-mediated immunotherapy is the expression of complement inhibitory proteins that are often upregulated on the tumor cell surface. It is hypothesized that the downregulation of complement inhibitors expressed on tumor cells will significantly enhance tumor sensitivity to antibody-mediated immunotherapy and may also enhance the outcome of a normally ineffective humoral immune response. Although frequently upregulated on tumor cells, complement inhibitory proteins remain largely unexplored as targets for cancer therapy due to their widespread expression. For some cancers, intracavitary delivery of a therapeutic reagent to modulate complement inhibitor function may obviate a requirement for specialized targeting strategies. It is proposed to investigate a therapeutic strategy involving intravesical (bladder) delivery of small interfering RNAs (siRNAs) to downregulate complement inhibitory proteins on bladder tumor cells, together with intravenous administration of a complement activating mAb directed against a bladder cancer-associated antigen. Optimum siRNA sequences for downregulation of complement inhibitory proteins on a mouse bladder cancer cell line will be determined in vitro. Selected siRNA sequences will be used to determine optimum strategy for siRNA/gene delivery to tumor cells in an orthotopic syngeneic mouse model of bladder cancer. Naked siRNAs and adenoviral vector delivery of siRNA will be investigated. Finally, the effect of complement inhibitor downregulation on the immune response to bladder cancer in the absence and presence of intravenously administered mAb immunotherapy will be investigated in an orthotopic mouse model.

Cerebral ischemia initiates a cascade of events that can lead to secondary neuronal damage resulting in an increased extent of infarct and poorer clinical outcome. The restoration of blood flow to the area surrounding the infarct, although essential for patient recovery, elicits an inflammatory response that plays a significant role in secondary injury. The complement system plays a key role in the pathophysiology of many inflammatory and ischemic diseases, and clinical studies and interventional studies in experimental models indicate an important role for complement in neuronal injury following ischemic stroke. The long term goal of this project is to gain a better understanding of complement-dependent mechanisms involved in the pathogenesis of ischemic stroke, and to develop an efficacious and safe neuroprotective strategy based on attenuating complement-dependent injury. We will characterize the effects of various novel complement inhibitors that target either complement activation products or selectins. The role and relative contributions of P and E selectin in the context of complement activation will be investigated by the use of selectin targeting moieties with function blocking activity. The targeting strategies have been validated in other models of inflammation and ischemia and reperfusion injury, and we have shown that appropriate targeting of complement inhibitors not only markedly increases their bioavailability and efficacy, but obviates the need to systemically inhibit complement. Systemic complement inhibition and immunosupression is a potential concern with regard to the treatment of stroke patients, since infectious complications occur in a high proportion of patients within the first few days after stroke. The targeted complement inhibitors will be characterized in a murine model of transient focal cerebral ischemia. In addition to therapeutic determinations, we will elucidate pathophysiological mechanisms by investigating the roles and contributions of each selectin and different complement activation products in cerebral ischemia and reperfusion injury. To this end, we will determine in vivo relationships between neuronal injury, functional recovery, local and systemic complement inhibition, the generation of different complement activation products, P vs. E selectin antagonisms with and without complement inhibition, cytokine production, adhesion molecule expression, leukocyte infiltration and activation, and platelet adhesion and aggregation.

[unreadable] DESCRIPTION (provided by applicant): Brain death (BD) and ischemia reperfusion injury (IRI) are unavoidable consequences of heart transplantation. Brain death produces profound physiologic derangements and the systemic effects of this central injury, although little studied, are known to contribute to peripheral organ ischemia, the upregulation of adhesion molecules, cytokine expression and leukocyte accumulation within the donor heart. Brain death induced inflammation in the donor also renders the heart more susceptible to IRI in the recipient, and there is evidence to indicate that both of these injurious events have negative long-term consequences with regard to allograft survival. Our working hypothesis is that complement plays a central role in causing myocardial BD- induced inflammation and injury, and enhances IRI in the recipient. We propose that a complement inhibitory strategy applied to the donor (in addition to the recipient) will provide protection from inflammation and injury, and as a consequence, will improve long-term graft survival due to decreased graft immunogenicity and host alloresponsiveness. We propose to utilize relevant mouse models to determine the role of complement in myocardial brain death induced injury (BDI) and in IRI following heart transplantation. Our investigations will focus on complement effector mechanisms and in vivo interactions between complement and P-selectin, an adhesion molecule that is strongly implicated in myocardial IRI and that is expressed in the heart following BD. We further propose to develop novel therapeutic strategies based on targeted complement inhibition and P-selectin antagonism, and to characterize the inhibitors in our newly developed mouse model of heart transplantation incorporating donor BD. We specifically propose to: 1. Determine complement effector mechanism(s) involved in myocardial injury following brain death 2. Develop and characterize novel therapeutic strategies based on P-selectin targeted complement inhibition and P-selectin antagonism and, 3. Determine the effect of anti-complement therapy in the BD donor, the recipient, or both, on the severity of myocardial IRI after heart transplantation and on the development of an alloimmune response and acute rejection of the graft. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Many conventional therapies of cancer, such as chemotherapy, radiotherapy and immunotherapy, depend to varying extents on the induction of tumor cell apoptosis. The effectiveness of apoptosis-inducing therapy can be significantly enhanced by an initial local inflammatory reaction at the tumor site and a subsequent specific immune response. Indeed, an inflammatory reaction following the induction of apoptosis has been shown to be important for the complete regression of some malignancies in rodent models. The complement system plays an important role in the inflammatory reaction and can modulate the development of both B and T cell responses. However, in the context of apoptosis, binding of natural IgM and complement activation and opsonization is critical for effective phagocytic uptake of apoptotic cells, a process considered important for limiting and resolving inflammation and for modulating immunity. We have shown that an immunotherapeutic approach involving targeted complement inhibition changes the inflammatory and immune profile within the tumor environment following radiotherapy, and significantly enhances therapeutic outcome in terms of modulating tumor growth, animal survival and induction of an anti-tumor T cell response. Our objective is to understand the mechanisms involved in this response, as well as to develop a novel strategy to enhance the therapeutic outcome of radiotherapy based on targeted complement inhibition. We hypothesize that the binding of self-reactive IgM to apoptotic tumor cells is involved in complement activation and opsonization of apoptotic cells, and that inhibiting complement-dependent apoptotic cell uptake after radiotherapy will promote necrosis, create an enhanced immuno-stimulatory environment, modulate macrophage and dendritic cell activation and differentiation, and augment or trigger specific immunity against a tumor. We will test the above hypothesis and investigate the role of IgM specificity and different complement opsonins using in vivo and in vitro therapeutic paradigms.

DESCRIPTION (provided by applicant): Ischemia/reperfusion injury (IRI) is a major clinical problem associated with several clinical conditions (e.g., hemorrhagic shock, severe burns, sepsis, myocardial infarction, stroke, transplant dysfunction/rejection). Complement is known to play a key role in sterile inflammation and injury following ischemia and reperfusion (IR), but more recently it has become evident that complement also plays important roles in tissue repair and regeneration following certain ischemic events. A goal of the proposed studies is to better understand the complement activation event following IR, and to dissect the dual role of complement effector mechanisms in IRI and tissue repair/regeneration. Concurrent with these goals, it is proposed to develop and characterize a novel targeted complement inhibitory strategy that protects against inflammation and injury while promoting tissue repair and regeneration. Previous and preliminary data indicate similar complement activation and effector mechanisms are involved in the injury and repair/regeneration processes of multiple organs and tissues. However, our model will be hepatic IRI and regeneration since mouse models have been well characterized and because of clinical significance. The overall working hypothesis is that complement is activated by natural self-reactive IgM that binds to neoepitopes exposed after ischemia, and that the terminal membrane attack complex (MAC) plays a key role in ischemia reperfusion injury (IRI) whereas more proximal activation products are important for repair and regeneration. We have isolated a panel of novel self-reactive IgM mAbs, and will use these mAbs to investigate immunological changes (neoepitope exposure) and the complement activation event that occurs after either IR or partial hepatectomy. We will also utilize complement inhibitors that function at different points in the cascade to investigate complement effector mechanisms involved in inflammation, injury and regeneration. We will also investigate the role of complement in phagocytic clearance of apoptotic and necrotic cells, and how this process impacts the inflammatory environment and subsequent tissue injury and repair. Finally, we will investigate how data generated in our mouse models relates to humans by analysis of clinical specimens. An additional goal of these studies is to develop a novel targeting strategy to locally deliver a complement inhibitor to post-ischemic neoepitopes utilizing single chain antibodies.

? DESCRIPTION (provided by applicant): Natural self-reactive IgM antibodies represent a class of innate pattern recognition receptors that recognize danger associated molecular patterns (DAMPS) as neoepitopes expressed on stressed or dying cells. Recognition of these neoepitopes by IgM activates complement, initiating an inflammatory reaction, which can have injurious as well as protective effects. The overall goals of this project are to better characterie the innate immunogenic alterations that occur following ischemia and stress, to fully characterize the neoepitopes that serve as DAMPs, and to understand the complement activation event and complement effector mechanisms involved in the balance between sterile inflammation and injury on the one hand, and tissue repair/regeneration on the other. Complement inhibitors will be used in mouse models as therapeutically relevant investigative tools, and the focus of the studies will be on hepatic ischemia reperfusion injury and regeneration.

? DESCRIPTION (provided by applicant): Ischemia reperfusion injury is an unavoidable event in organ transplantation, and it is a major clinical problem that is thought to play an important role in both short-term and long-term graft survival. The mechanisms responsible for induction of post-surgical inflammation and ischemia reperfusion injury are not well defined, although complement, and more recently natural self-reactive IgM, have been shown to play key roles in the process. We have identified neoepitopes that become expressed in cardiac grafts, and which bind natural IgM. We have prepared constructs that target complement inhibitors to a particular neoepitope, and we propose to use these constructs to study the role of IgM in immune sensing of cardiac injury and the subsequent activation of complement. Using mouse models of cardiac transplantation, as well as a series of in vitro model systems, we will investigate how various complement activation products modulate an alloimmune response and acute rejection. We will also investigate how different neoepitopes and their recognition in cardiac grafts relate to each other, and how the length of ischemia and extent of ischemia reperfusion injury relates to neoepitope expression and the priming of an alloimmune response.

Recombinant tissue-type plasminogen activator (tPA) is currently the only approved pharmacological agent for the treatment of ischemic stroke, and in general it must be administered within 4.5 hours of symptom onset. Because of this time constraint, as well as dangers associated with this drug, it is estimated that only about 5- 10% of stroke patients are treated with tPA. There is thus a significant need for new and effective approaches to treat stroke. It is now clear that the complement system plays an important role in the propagation of inflammation and injury following cerebral ischemia and reperfusion (ischemic stroke), and the overall goal of this project is to develop and characterize a novel, effective and safe strategy of site-targeted complement inhibition that can be applied several hours after stroke, and that will improve long-term cognitive and functional recovery. To identify the most effective complement inhibitory approach, we will prepare and characterize various complement inhibitors that block different parts of the complement pathway and that are targeted to the site of ischemic brain injury via a novel approach. The constructs will be investigated using a mouse model of middle cerebral artery occlusion and reperfusion, and we will investigate the effect of our constructs on cerebral injury, repair and neuroregeneration, and acute and chronic cognitive and motor function outcomes. Following identification of the optimum type of complement inhibitory construct, we will complete a series of pre-clinical determinations that will assist in future drug development. These studies will include PK and PD determinations, dose response, treatment window, dosing schedule, and effect in young and aged mice (since the risk of stroke increases with age). Finally, we will investigate our strategy of complement inhibition in the context of tPA therapy to determine whether there are any adverse effects associated with co-administration of the two pharmacological reagents.

Abstract In the context of heart transplantation (Tx), which is the focus of this application, primary graft failure and cardiac allograft vasculopathy (AV) remain the major limitations to short and long-term survival. The course, severity and onset of AV have changed little since the inception of cardiac Tx surgery, despite improvements in T cell immunosuppression. The precise mechanisms involved in the development of primary graft failure and chronic AV are not well understood, here we investigate the role of donor brain death (BD)-induced injury and ischemia reperfusion injury (IRI) in the development of AV, and the role of complement in these processes. Brain death-induced injury and IRI are unavoidable events in most organ transplantations, and they are major clinical problems that are thought to play important roles in both short-term and long-term graft survival. Natural self-reactive IgM and complement play a major role in both types of injury, and here we investigate novel approaches of using graft-targeted IgM blockade and complement inhibition, administered as an acute immunosuppressant after cardiac transplantation, to determine how acute post-transplant inflammation and injury modulates the development of AV (chronic rejection). We will also investigate how complement and graft-targeted strategies of IgM and complement inhibition affect the development of cardiac AV in the context of subtherapeutic T cell immunosuppressive therapy (rapamycin and tacrolimus), and how the use of complement inhibition as an adjuvant therapy modulates AV. By linking moieties that target injury-specific post- ischemic neoepitopes expressed in cardiac (and other organ/tissue) grafts to different complement inhibitors, we will be able to investigate how complement modulates acute injury and subsequent chronic rejection. We expect to elucidate complement-dependent mechanisms involved in the development of AV, and to identify a therapeutic candidate for further development that will permit the use of immune-sparing immunosuppressive therapies.

PROJECT SUMMARY / ABSTRACT    This new application from the Medical University of South Carolina (MUSC) seeks support for a Program  in  Immunology  Research  and  Entrepreneurship  (PIRE)  to  train  postdoctoral  fellows  in  immunology  with  an  emphasis on translation, biomedical innovation and entrepreneurship. The program seeks to address the need  to  empower  our  future  immunology  research  workforce  with  the  competence  necessary  to  commercialize  scientific discoveries. Disease-­related themes that are central to this program are innate immune mechanisms,  immunity  to  infection,  alloimmunity  (organ  and  hematopoietic  stem  cell  transplant),  autoimmunity,  and  cancer  immunity. The Program Mentor Faculty consist of an interdisciplinary group of 14 investigators with established  training records who are committed to research and research training of postdoctoral trainees within the area of  inflammation and immunity. The proposed grant will support 2 fellows in the first year and 4 per year thereafter  to standardize recruitment efforts and provide a steady flow of new trainees. The core training for PIRE fellows  is the research experience in the laboratory setting complemented by specialized training in entrepreneurship,  using the following components: (1) mentoring through the engagement of an Individual Development Plan for  Entrepreneurship  (IDPE)  Committee;?  (2)  laboratory  training  including  use  of  mass  cytometry,  a  potentially  transforming technology for cellular immunology research;? (3) didactic training in biomedical commercialization  and  entrepreneurship;?  (4)  experiential  training  by  conducting  an  internship  with  MUSC?s  technology  transfer  office;? (5) participation in activities (seminar series, journal club, scientific retreat) sponsored by the Department  of Microbiology and Immunology;? (6) instruction in the responsible conduct of research;? (7) instruction in methods  for enhancing reproducibility;? and (8) use of a virtual ?trainee career toolkit? that includes grantsmanship, teaching  and career planning. Recruitment of postdoctoral trainees will be carried out on a national basis with an explicit  goal of recruiting women and minority postdoctoral trainees to address significant disparities in entrepreneurial  participation  as  well  as  biomedical  and  immunology  research.  The  Program  Director  will  be  assisted  by  an  Associate  Program  Director,  administrative  coordinator,  Program  Steering  Committee  and  External  Advisory  Board to ensure effective administration, management, and evaluation of the program and guide future direction. 

Abstract Muscle loss due to trauma, tumor resection or congenital malformation is devastating to the patient and their family. Current treatment regimens involve creative rearrangement of skin and muscle flaps to mitigate the deformity. However, these approaches are often sub-optimal as a percentage of mobilized flap tissues contract, atrophy and/or die and do not function like the original tissues. The field has long envisioned a treatment modality where using the patient's own cells to create tissue grafts, that can then be transplanted into a patient to restore form and function. However, unfortunately, there are numerous hurdles yet to be overcome for this new treatment plan to be broadly adopted in the clinic. One urgent and underappreciated hurdle is the poor survival rate of implanted cells which we hypothesize are killed by the innate immune response to the implanted graft. A major effector mechanism of innate immunity is the complement (Cp) system. The extent that early Cp inflammatory events kill a substantial number of cells within the TECs remains an important unanswered question in the tissue- engineering field. Even if cell death due to early inflammation can be avoided, there is insufficient vascular support. Techniques to generate pre-vascularized TECs which have rapid anastomotic potential would substantially improve cell survival and engraftment. These two highlighted events are intimately linked. Reduction of early inflammation without early vascular support or early vascular support in the presence of a substantial inflammatory response will still result in cell death and failed repair. Therefore, our central hypothesis is that; modulation of Cp effector pathways will promote survival of TEC by reducing inflammation, direct cell injury, and by promoting neovascularity. In this proposal we articulate a line of investigation that will lead to an enabling technology to improve the engraftment of cellularized implants by modulating the early inflammatory process and promoting nascent microvascular beds. Our lab has invented novel strategies and therapeutics that target the early inflammatory response. Additionally, we have recently developed a novel scaffold-free technology to generate Self-organizing Pre-vascularized Endothelial-fibroblast Constructs (SPECs). These engineered constructs can become perfused with blood very quickly once implanted. Using known mechanisms of inflammation, angiogenesis and anastomosis as our guide, we will systematically test these innovative technologies using specific inhibitors and genetically engineered mice in a sub-muscular implant model. The expected outcomes would significantly move the field forward by providing a workable solution to the principal hurdles facing tissue engineering - rapid vascularization and survival of implanted cells and tissues.