Kyra J. Becker, MD - US grants

The hypotheses to be tested in this proposal are that the post-ischemic immune response contributes to brain injury and that brain injury can be reduced by appropriately modulating the immune response. Soon after the onset of ischemic stroke, lymphocytes infiltrate the brain and an immune response is triggered against brain antigens. Using an animal model of transient middle cerebral artery occlusion, we will define the nature of the post-ischemic immune response and explore ways to manipulate that response for therapeutic benefit. The specific aims of the project are: 1) to determine the time between ischemic stroke onset and development of a cellular immune response directed towards brain antigens, and to analyze that response, 2) to determine the nature and time course of lymphocyte infiltration into brain after stroke, and 3) to define the therapeutic potential of modulating the post-ischemic cellular immune response.

The hypotheses to be tested in this proposal are that the post-ischemic immune response contributes to brain injury and that brain injury can be reduced by appropriately modulating the immune response. Soon after the onset of ischemic stroke, lymphocytes infiltrate the brain and an immune response is triggered against brain antigens. Using an animal model of transient middle cerebral artery occlusion, we will define the nature of the post-ischemic immune response and explore ways to manipulate that response for therapeutic benefit. The specific aims of the project are: 1) to determine the time between ischemic stroke onset and development of a cellular immune response directed towards brain antigens, and to analyze that response, 2) to determine the nature and time course of lymphocyte infiltration into brain after stroke, and 3) to define the therapeutic potential of modulating the post-ischemic cellular immune response.

DESCRIPTION (provided by applicant): The primary aims of this study are to determine how many patients become sensitized (develop a Th1 immune response) to brain antigens after stroke and whether infection in the immediate post-stroke period increases the risk of becoming sensitized to those antigens. The rationale for this study is based on the fact that the integrity of the blood-brain barrier is breached in stroke; cells of the immune system thus encounter novel central nervous system (CMS) antigens in both the brain and in the systemic circulation. This encounter may result in an immune response to those antigens and the microenvironment at the site of encounter determines the nature of the immune response generated. For instance, a systemic inflammatory response, such as occurs with infection, could induce the expression of costimulatory molecules and promote sensitization of lymphocytes (Th1 immune response) to brain antigens. In animal models of stroke, lymphocytes sensitized to CMS antigens contribute to cerebral injury and manipulation of the immune response improves outcome from stroke. Similar manipulation of the immune response could provide a therapeutic target for clinical intervention. To date, however, attempts at manipulating the immune response in patients with stroke have produced either no clinical benefit or even harm. Thus, prior to conducting further trials of immune modulation in clinical stroke, the nature and the consequences of the post-ischemic immune response need to be understood. For the purposes of this study, antigen-specific immune responses to brain antigens will be evaluated serially over the course of 1 year in patients who present with acute ischemic stroke; the type of immune response, Th1 versus Th2/Th3, will be compared between patients who develop infection in the immediate post-stroke period and those who do not. The effect of stroke subtype and endogenous immunomodulatory responses on the likelihood of becoming sensitized to brain antigens will also be assessed. Progression of white matter disease and brain atrophy, as detected by magnetic resonance imaging, will be used as a surrogate measure of the pathologic consequences of a Th1 response. Data derived from this study will be used to plan future trials of immunomodulation in patients with stroke.

Stroke is a leading cause of disability in developed countries. At least 25% of persons who suffer stroke will become infected during the course of their acute stroke, and those who become infected experience more disability than those that remain infection free. How systemic infection contributes to neurologic injury during stroke is not well understood. The rationale for the experiments outlined in this proposal is based on the fact that there is a breach in the integrity of the blood-brain barrier (BBB) following stroke that allows the immune system to encounter novel central nervous system (CNS) antigens in both the brain and in peripheral lymphoid organs. The type of immune response generated upon antigen encounter is determined by the composition of the microenvironment at the site of the encounter. The systemic inflammatory response that accompanies an infection could induce the expression ofcostimulatory molecules and alter the context in which antigens are presented to lymphocytes, thus promoting their sensitization to brain antigens. Preliminary data suggestthat animals do develop an autoimmune response to brain following stroke and that lymphocytes sensitized to CNS antigens contribute to cerebral injury, which might explain why infection in the post-stroke period is associated with worse outcome. Manipulating the post-ischemic immune response could therefore be an effective therapeutic intervention for the treatment of stroke. Using an animal model of stroke and a number of in vivo and ex vivo immunologic assays,we plan to confirm and extend our prior findings which show that sensitization to CNS is associated with worse outcome after stroke. The proposed studies will also incorporate sensitive measures of neurological and behavioral performance. More importantly, we hope to show that induction of immunologic tolerance to CNS antigens, even after stroke onset, will prevent the development of CNS autoimmunity; thistolerance should translate to improved outcome from stroke. Given that over 700,000 strokes occur each year in the United States and that at least 175,000 of patients with stroke will develop a concomitant infection, an immune modulating therapy could have significant impact on public health.

DESCRIPTION (provided by applicant): This proposal is a competing renewal which aims to better understand why infection, which is common after stroke, negatively impacts outcome. Our research demonstrates that an immune response to central nervous system (CNS) antigens occurs after stroke and that the nature of this immune response depends upon the experimental conditions and affects neurological outcome. Specifically, Lewis rats that develop a TH1 type immune response to brain antigens experience worse outcome while Lewis rats that develop a regulatory type response (TREG) experience better outcome. The predisposition to developing a TH1 response is increased by systemic inflammation induced with lipopolysaccharide (LPS). We believe that this model may help to explain why patients who develop an infection following stroke experience worse outcome. The goals of the current project are to assess the clinical relevance of our model by validating our findings using a true infection model. We further aim to better define the nature and regulation of this post-ischemic immune response and assess the effect of antibiotic therapy and the role of the sympathetic nervous system on this response. The primary hypothesis to be addressed in the research outlined is: does systemic infection in the immediate post-stroke period increase the likelihood of developing a detrimental autoimmune response to CNS antigens?

? DESCRIPTION (provided by applicant): The objective of the proposed project is to investigate the potential of the gasotransmitter carbon monoxide (CO) as a neuroprotective agent in acute ischemic stroke (AIS) using a novel oral formulation of CO (HBI-002). Numerous studies, both in vitro and in vivo, demonstrate that CO has cytoprotective properties through anti- oxidant, anti-inflammatory and anti-apoptotic processes. In five studies in three independent laboratories, researchers found that the heme-oxygenase (HO)-1/CO pathway provides neuroprotection in animal models of AIS. In four of these studies exogenous administration of CO, which has been shown to up-regulate HO-1, reduced cerebral infarct size, improved behavioral scores and increased blood flow to the infarct border zone in experimental stroke. These independent studies provide compelling support for a potential beneficial role for the use of CO in the treatment of AIS. However, CO administration strategies have been limited to inhalation and intravenous approaches that carry inherent risks of safety and toxicity. HBI-002, an aqueous carboxylipid-protein liquid formulation, is being developed for the treatment of AIS. The administration of a defined dose of CO delivered by oral administration of HBI-002 obviates the problems associated with previously studied inhaled or intravenously administered carrier-metal CO. HBI-002 comprises a water-based solution containing CO. Proof of concept manufacture of HBI-002 has been demonstrated. Pharmacokinetic and pharmacodynamic studies in rats and in two adult healthy volunteers have demonstrated proof of concept feasibility, tolerability, and bioavailability. The next step in development is to demonstrate that CO delivered via the oral administration of HBI-002 improves outcome in appropriate AIS animal models and to further understand the potential mechanisms of neuroprotection.