Protective Mechanisms In Cerebral Ischemia

脑缺血的保护机制

基本信息

批准号：
9358541
负责人：
John Hallenbeck
金额：
$ 100.79万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9358541
关键词：
Animals Antibodies Astrocytes Atherosclerosis Attenuated Baculoviruses Binding Brain Brain Injuries Brain Ischemia Cell Adhesion Molecules Cells Cerebral Ischemia Clinical Trials Complement 3a Consensus Critical Pathways Cytoprotection DNA Delayed Hypersensitivity Deposition Dose E-Selectin E1A-associated p300 protein Elements Endothelial Cells Endothelium Experimental Autoimmune Encephalomyelitis Genes Glucose Goals Hemorrhage Homeostasis Hour Human Hypoxia ITGAM gene Immune Immunotoxicology Impairment Inbred SHR Rats Infarction Inflammatory Injection of therapeutic agent Intercellular adhesion molecule 1 Investigational Drugs Ischemia Ischemic Stroke Knockout Mice L-Selectin Lipids Lipopolysaccharides Maximum Tolerated Dose Measures Mediating Mediator of activation protein Middle Cerebral Artery Occlusion Modeling Molecular Monoclonal Antibodies Mus NF-kappa B Neurons New Mexico Nuclear Translocation Oxygen P-Selectin Perfusion Phase Phase I Clinical Trials Phosphorylation Play Pre-Clinical Model Proteolysis Rare Diseases Rattus Recombinants Regulatory T-Lymphocyte Research Research Project Grants Role SOD2 gene Safety Secondary Prevention Site Splenocyte Sprague-Dawley Rats Stress Stroke Stroke prevention Subarachnoid Hemorrhage Subcortical Leukoencephalopathy TNF gene Testing Thrombomodulin Toxicology Translating Universities Up-Regulation Vascular Cell Adhesion Molecule-1 Vascular Dementia Vasospasm Work acute stroke adaptive immunity adverse outcome aortic arch bench to bedside deprivation enlimomab expression vector glycosylation granulocyte humanized mouse in vitro Model in vivo nonhuman primate novel strategies open label p65 pre-clinical preconditioning prevent response stroke treatment tumor necrosis factor binding protein von Willebrand Factor western diet white matter damage

项目摘要

Molecular and cellular mediators of innate and adaptive immune responses play a major role in both the initiation and the progression of stroke. They also participate in the induction of tolerance to ischemia by sublethal preconditioning stresses. We have investigated such mediators in the three abovementoned facets of stroke. Inhibition of tumor necrosis factor(TNF) with TNF-binding protein reduces brain infarct volume in middle cerebral artery occlusion (MCAO) models in the rat and mouse. In addition, TNF-binding protein attenuates the progressive impairment of microvascular perfusion that occurs during the early hours of focal brain ischemia. These findings implicate TNF as a mediator of progressive brain damage during acute stroke. Lipopolysaccharide (LPS) pretreatment has been demonstrated to induce tolerance to focal brain ischemia in the MCAO model in spontaneously hypertensive rats (SHR). TNF binding protein blocks this tolerance implicating TNF as a mediator of this state of preserved homeostasis under stress. This form of tolerance also reduces the degree of microcirculatory perfusion impairment in brain. Preconditioning with TNF by intracisternal injection of TNF induces tolerance to ischemia in the Balb/C mouse. In vitro models comprising cellular elements of brain have been established in order to examine the mechanisms involved in the observed in vivo tolerance to ischemia of the brain pretreated either by TNF or by oxygen/glucose deprivation (OGD). Pretreatment of primary neuronal cultures with short hypoxia (15 minutes) 24 hours prior to 60 minutes of hypoxia, protected neurons against hypoxia (number of dead cells was 9.4% versus 35% in non-pretreated cultures). Pretreatment with TNF (50 ng/ml) also protected cortical neurons against 60 minutes of hypoxia. TNF-preconditioning can induce tolerance to subsequent OGD in brain microvessel endothelial cells, astrocytes and cortical neurons. In cortical astrocytes from 2-3 day old Sprague-Dawley rats, preconditioning with TNF-alpha to produce tolerance does not inhibit I-kappaB proteolysis, nuclear translocation of NF-kappaB or binding of the p65 subunit of NFkB to its consensus site on DNA. It does, however, prevent p65 phosphorylation and consequently disrupts the association of the coactivator protein, p300/CBP, with that subunit. The result is that expression of proinflammatory genes such as ICAM-1 is inhibited, but expression of cytoprotective genes such as manganese superoxide dismutase continues unabated. In bedside to bench studies, mouse anti-rat ICAM-1 antibody induced an inflammatory state in preclinical models of ischemic stroke that included activation of complement (C3a desArginine), granulocytes (CD11b up-regulation), and endothelium (E- and P-selectin expression). Serial administration of the antibody sensitized rats to produce anti-mouse antibodies and augmented infarct size in a focal brain ischemia model. Similar responses to the mouse anti-human ICAM-1 monoclonal antibody (a non-humanized antibody), Enlimomab, may have contributed to the adverse outcomes of the Enlimomab acute stroke trial. A form of immunological tolerization, mucosal tolerance, has been shown to target regulatory T cells to activating endothelium and to prevent strokes. Mucosal tolerance to E-selectin, which is an adhesion molecule that only becomes expressed on endothelium when a vessel segment becomes activated, has been shown to profoundly reduce ischemic strokes and to eliminate hemorrhages that otherwise occur spontaneously in spontaneously hypertensive and genetically stroke-prone rats (SHR-SP). E-selectin tolerization also can reduce infarct size after MCAO in SHR-SP and this cytoprotection can be adoptively transferred by splenocytes from tolerized animals indicating that the protection is cell-mediated. This novel approach to stroke prevention is being translated into clinical trials involving the secondary preventon of stroke and Binswanger Disease. GMP recombinant murine and human E-selectin has been produced with a baculovirus expression vector platform and is being used in preclinical toxicology and immunotoxicology studies in mice and non-human primates as a prerequisite for obtaining an IND. Additional studies have shown that E-selectin tolerization markedly inhibits white matter damage in models of vascular dementia and experimental autoimmune encephalomyelitis. E-selectin tolerization also suppresses delayed vasospasm in a subarachnoid hemorrhage model. A bedside to bench proposal has supported the study of E-selectin tolerization in a model of atherosclerosis where the tolerization suppresses lipid deposition in the aortic arches of APOE null mice on a Western diet. Current work follows a critical path to Phase I testing. We are currently working on a "go-no go" test in which we need to observe suppression of delayed type sensitivity in "humanized" mice in order to proceed. If successful, we plan to file an IND and initiate a Phase I clinical trial in Binswanger Disease, an Orphan Disease, with Dr. Gary Rosenberg at the University of New Mexico. If the initial trial raises no flags, we will continue with the Binswanger studies at U. New Mexico.

先天和适应性免疫反应的分子和细胞介质在中风的起始和进展中都起着重要作用。他们还通过共生的预处理应力来参与对缺血的耐受性。我们已经在中风的三个方面进行了调查。用TNF结合蛋白抑制肿瘤坏死因子（TNF）可减少大鼠和小鼠中脑动脉闭塞（MCAO）模型中脑梗塞体积。此外，TNF结合蛋白减弱了在局灶性脑部缺血早期发生的微血管灌注的进行性损害。这些发现暗示TNF是急性中风期间进行性脑损伤的介体。已证明脂多糖（LPS）预处理可诱导自发性高血压大鼠（SHR）中MCAO模型中对局灶性脑缺血的耐受性。 TNF结合蛋白阻止了这种耐受性，这意味着TNF是在压力下保存的稳态状态的介体。这种耐受性也降低了大脑中微循环灌注障碍的程度。通过内注射TNF的TNF进行预处理，可诱导BALB/C小鼠缺血的耐受性。已经建立了包含大脑细胞元素的体外模型，以检查与TNF或通过氧气/葡萄糖剥夺（OGD）预处理的体内耐缺血性耐受性相关的机制。在60分钟缺氧前24小时预处理缺氧（15分钟）的原发性神经元培养物，保护神经元免受缺氧的保护（死细胞的数量为9.4％，而在未经培养的培养物中为35％）。用TNF（50 ng/mL）预处理还保护皮质神经元可抵抗60分钟的缺氧。 TNF前提可以诱导对随后的OGD的耐受性，在脑微血管内皮细胞，星形胶质细胞和皮质神经元中。在2-3天大的Sprague-Dawley大鼠的皮质星形胶质细胞中，用TNF-Alpha进行预处理以产生耐受性不会抑制I-kappab蛋白水解，NF-kappab的核易位或NFKB p65亚基与其在DNA上的共有点的p65亚基的结合。但是，它确实可以防止p65磷酸化，因此破坏了共激活因子p300/cbp与该亚基的关联。结果是抑制了促炎基因（例如ICAM-1）的表达，但是细胞保护基因（例如锰超氧化物歧化酶）的表达却没有延续。在基准研究的床边，小鼠抗抗rat ICAM-1抗体在缺血性中风的临床前模型中诱导了炎症态，其中包括补体的激活（C3A去骨氨酸），粒细胞（CD11B上调）和内皮（E-和P-系列素表达）。抗体敏化大鼠的序列给药以产生抗小鼠抗体并增加局灶性脑缺血模型中的梗死大小。对小鼠抗人ICAM-1单克隆抗体（一种非人性化抗体）ENERIMOMAB的反应可能导致Enlimomab急性中风试验的不良结果。一种免疫学耐受性，粘膜耐受性的一种形式已被证明靶向调节性T细胞激活内皮并预防中风。粘膜对E-选择素的耐受性，这是一种粘附分子，仅当血管段被激活时才在内皮上表达，已被证明可深刻地减少缺血性中风，并消除自发性高度高度和基因性疾病中的大鼠（SHR-SHR-SHR-SHR）自发地自发地发生的出血。 E-选择蛋白的耐受性也可以减少SHR-SPH中MCAO后的梗塞大小，并且可以通过脾细胞从耐受动物的脾细胞转移这种细胞保护，这表明该保护是细胞介导的。这种预防中风的新方法正在转化为涉及中风和BINSWANGER疾病的次要预防的临床试验。 GMP重组鼠和人E-选择蛋白已经用杆状病毒表达载体平台产生，并用于小鼠和非人类灵长类动物的临床前毒理学和免疫毒性研究中，作为获得IND的先决条件。其他研究表明，在血管痴呆和实验性自身免疫性脑脊髓炎模型中，E-选择蛋白耐受明显抑制了白质损害。 E-选择蛋白的耐受性还抑制了蛛网膜下腔出血模型中的延迟血管痉挛。基准提案的床头支持在动脉粥样硬化模型中研究E-选择蛋白耐受性的研究，其中耐受化抑制了西方饮食中Apoe Null小鼠的主动脉弓中的脂质沉积。当前的工作遵循了I期测试的关键途径。我们目前正在进行一项“ GO-NO GO”测试，其中我们需要观察“人性化”小鼠中延迟类型敏感性的抑制。如果成功，我们计划提交IND，并与新墨西哥大学的Gary Rosenberg博士一起在Binswanger病，一种孤儿病的I期临床试验。如果最初的试验不提出旗帜，我们将继续在美国新墨西哥州进行Binswanger研究。