Pharmacology Of Neurotoxins

据报道，在慢性HIV感染和各种类型的脑损伤后，激活的巨噬细胞/小胶质细胞释放了可溶性神经毒素和促生长因子。利用新生大鼠的神经元培养细胞生物测定进行了改进以进行定量测量。用该生物测定方法对从活化的人外周血单核细胞或HIV感染的巨噬细胞的条件培养液中分离出的可溶性因子进行了监测。条件培养液的提取物被分成不同的成分类别(脂类与非脂类，低相对分子质量与高相对分子质量)。在11批来自HIV感染-内毒素刺激的巨噬细胞的条件培养液中，有6批显示出神经元毒性。在含有小于3000Da物质的无脂部分中，酶活性最高。神经毒性为胰酶不敏感。谷氨酸浓度在神经毒性介质中升高。三项测试表明，仅谷氨酸浓度就足以解释观察到的神经毒性。谷氨酸浓度降至对照水平，条件培养液中加入谷丙转氨酶可消除神经毒性。预先与星形胶质细胞孵育的神经毒性介质也降低了谷氨酸浓度，并消除了神经毒性。最后，将谷氨酸添加到对照条件培养液中，其浓度与神经毒性HIV/LPS培养液中的浓度相匹配，复制了观察到的神经毒性效应的程度和形态。另外两项神经毒理学调查正在进行中。首先，我们正在验证一种假设，即炎症后外周细胞重新聚集到中枢神经系统是由白三烯介导的。在沙土鼠和大鼠身上，使用含有免疫刺激剂(如脂多糖或兴奋性氨基酸)的立体定向微量注射溶液，研究了中枢神经系统炎症后的生化事件。之前，我们在沙土鼠身上量化了色氨酸代谢增加的现象，作为免疫介导的人类神经疾病的特征。沙土鼠表现出与人类相同的生化反应，为研究这一途径在动物模型中的作用提供了一种手段。白三烯受体拮抗剂和脂氧合酶抑制剂的活性将被研究，以确定是否可以通过减少神经变性来改善由于外周血细胞浸润而造成的继发性损害。在第二项研究中，我们正在检验这一假设，即随着干细胞表型的发展，存在特定的蛋白质组变化。研究干细胞的分化过程可以阐明正常人脑发育过程中发生的复杂事件。大鼠骨髓基质细胞(BMSCs)是一种具有自我更新功能的多能干细胞，具有广阔的应用前景。在培养条件下，BMSCs可以被诱导为表达神经元标志蛋白的神经元表型。神经元表型表达与终末分化平行，使细胞分裂停止。这些研究的目的是描绘具有表型变化特征的核蛋白质组变化。来自基质细胞的DNA合成体和核蛋白将使用二维凝胶和质谱学技术进行表征。

The release of soluble neurotoxins and growth promoting factors by activated macrophages/microglia has been reported following chronic HIV infection and various types of brain injury. A neuronal cultured cell bioassay using neonatal rat was refined for quantitative measurements. The isolation and characterization of soluble factors isolated from conditioned media from activated human peripheral blood monocytes or HIV-infected macrophages was monitored with this bioassay. Extracts of conditioned media were separated into component classes (lipids vs. non-lipids, low versus high molecular weight). Neuronal toxicity was demonstrated in six of eleven batches of conditioned media from HIV-infected-LPS-stimulated macrophages. Activity was highest in a lipid-free fraction containing substances less than 3000 Da. The neurotoxicity was trypsin insensitive. Glutamate concentrations were elevated in neurotoxic media. Three tests demonstrated that glutamate concentrations are alone sufficient to account for the observed neurotoxicity. Glutamate concentrations were lowered to control levels and neurotoxicity was abolished by treatment of the conditioned media with glutamate pyruvate transaminase. Pretreatment of neurotoxic media by pre-incubation with astrocytes also reduced glutamate concentrations and abolished neurotoxicity. Finally, addition of glutamate to control conditioned media in concentrations matching those in the neurotoxic HIV/LPS media replicated the observed extent and morphology of the neurotoxic effect. Two other neurotoxicology investigations have are in progress. In the first, we are testing the hypothesis that recruitment of peripheral cells into the CNS following inflammation is mediated by leukotrienes. Biochemical events following CNS inflammation are been studied in gerbils and rats using stereotaxically micro-injected solutions containing an immuno-stimulant such as lipopolysaccharide or an excitatory amino acid. Previously, we quantified the phenomenon of increased tryptophan metabolism as characteristic of immune mediated human neurologic disease in gerbils. Gerbils exhibit the same biochemical response as humans, providing a means to study the role of this pathway in an animal model. The activity of leukotriene receptor antagonists and lipoxygenase inhibitors will be studied to determine whether the secondary damage due to peripheral blood cell infiltration can be ameliorated with reduction of neurodegeneration. In a second study, we are testing the hypothesis that there are specific proteome changes accompanying stem cell phenotype development. Investigating the differentiation process of stem cells could elucidate the complex events that occur during normal human brain development. Rat bone marrow stromal cells (BMSCs) are multipotent stem cells that are self-renewing with broad potential. When grown in culture, BMSCs can be induced to a neuronal phenotype expressing neuronal marker proteins. Neuronal phenotype expression parallels terminal differentiation, halting cell division. The objective of these studies is to profile nuclear proteome changes characteristic of the phenotypic changes. The DNA synthesomes and nuclear proteins from the stromal cells will be characterized using two-dimensional gel and mass spectrometric techniques.