Involvement Of Human Viruses Associated With Chronic Neurologic Disease

与慢性神经系统疾病相关的人类病毒的参与

• 批准号: 7969541
• 负责人: Steven Jacobson
• 金额: $ 209.83万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 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/7969541
• 关键词: Affect; Allogenic; Antibodies; Antigens; Antiviral Agents; Astrocytes; Attention; Autoimmune Diseases; Biological Assay; Bone Marrow Transplantation; Brain; CD14 gene; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Chronic; Clinical; Clinical Trials; DNA; Daclizumab; Data; Detection; Disease; Effector Cell; Encephalitis; Enzyme-Linked Immunosorbent Assay; Epilepsy; Etiology; Excision; Exhibits; Fatal Outcome; Foxes; Genes; Glutamates; Herpesviridae; Human; Human Herpesvirus 4; Human Herpesvirus 6; Human T-lymphotropic virus 1; Human Virus; IL2RA gene; Immune Tolerance; Immune response; Immunoglobulin G; In Vitro; Individual; Infection; Inflammatory; Interferon Type II; Interleukin 2 Receptor; Interleukin-15; JC Virus; Lead; Link; Lymphocyte; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Multiple Sclerosis; Nerve Degeneration; Neuraxis; Neuroglia; Neurologic; Pathogenesis; Patients; Pharmaceutical Preparations; Play; Population; Prevention; Production; Proteomics; RNA; Reporting; Research; Role; Satellite Viruses; Self Tolerance; Serum; Severities; Signal Pathway; Signal Transduction; Spastic Paraparesis; Spinal Cord; Spinal Cord Diseases; System; T-Lymphocyte; T-Lymphocyte Subsets; Taxes; Temporal Lobe Epilepsy; Testing; Time; Transactivation; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Tropical Spastic Paraparesis; Tysabri; Viral; Virus; Virus Activation; Virus Diseases; Work; chronic neurologic disease; cytotoxic; forkhead protein; macrophage; natalizumab; nervous system disorder; novel; peripheral blood; protein expression; protein profiling; skin lesion; surface enhanced laser desorption ionization; tax Genes; treatment strategy; uptake; virus host interaction; virus tropism

The human T lymphotropic virus type I (HTLV-I) is the etiologic agent of a chronic progressive myelopathy known as HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease clinically similar to the chronic progressive form of multiple sclerosis (MS). An understanding of the pathogenesis of a neurologic disease with a known viral etiology (HTLV-I and HAM/TSP) will aid in defining similar mechanisms of pathogenesis in MS, a disease of unknown etiology in which viruses have been suspected to play a role. Additionally, other viruses have also been associated with MS and particular attention has recently focused on ubiquitous human herpesvirus (HHV-6). Investigative research on this agent in MS has led to the study of other disorders that may also be associated with viruses including epilepsy, neurological complications following allogeneic bone marrow transplant, and encephalitis. The major findings of these studies have been: 1) Increased HTLV-I specific CD8+ cells have been shown to be elevated in the peripheral blood and CSF of HAM/TSP patients and directly proportional to the amount of HTLV-I proviral DNA and RNA. These antigen-specific T cells are considered to be immunopathogenic and may be directly involved in virus-host interactions in the CNS. 2) Direct observations of these antigen-specific CD8+ T cells in spinal cord sections of HAM/TSP together with HTLV-I infected CD4+ cells have been demonstrated and support a role for these cyotoxic effector cells in the pathogenesis of this disorder. 3) As another marker of cytoxicity, we demonstrated that spontaneous degranulation and IFN-gamma production (defined by CD107+ IFN-gamma+ cells) was correlated with proviral DNA load in CD14+ macrophages from HTLV-I-infected patients, and that enhanced IL-15 expression on CD14+ cells mediated the dysregulation of CD8+ T cells in HAM/TSP patients. The observation of HTLV-I infected macrophages is novel and suggests a wider tropism for this virus than previously reported. The effect of HTLV-I on CD14+ macrophages is also being investigated. 3) CD4+CD25+ regulatory T cells are important in the maintenance of immunological self-tolerance and in the prevention of autoimmune diseases. We have demonstrated that in HTLV-I infected CD4+CD25+ T cells of patients with HAM/TSP the expression of the forkhead transcription factor Foxp3, a specific marker of regulatory T cells, was lower than that of healthy individuals. We have shown reduced protein expression of Fox-P3 positive T regulatory cells in patients with HAM/TSP. We also have demonstrated that HTLV-I tax had a direct inhibitory effect for the Foxp3 expression and inhibited the regulatory function of these cells. These results suggest that direct human retroviral infection of CD4+CD25+ T cells may be associated with the pathogenesis of HTLV-I associated neurologic disease through the dysregulation of CD4+CD25+ regulatory T cells. 4) We have also demonstrated that the HTLV-I tax gene inhibits expression of Foxp3 by a mechanism that blocks TGF-beta signalling. Since TGF-b signalling has been shown to be critical for both Foxp3 expression and Treg function, we have also shown that this signalling pathway was dysregulated in HAM/TSP patients. We found that levels of Smad7 (a TGF-beta-inducible gene) and TGF-beta receptor II (TGF-bRII) in CD4+ T cells were significantly reduced in patients with HAM/TSP compared to normal donors. Furthermore, expression of TGF-bRII inversely correlated with the proviral load. Importantly, CD4+CD25+ and CD4+CD25- T cells from HAM/TSP patients exhibited reduced TGF-bRII expression compared to normal donors, which was associated with functional deficits in vitro, including loss of Treg suppressor function and escape of effector T cells from Treg-mediated control. This evidence suggests that a virus-induced breakdown of immune tolerance affecting both regulatory and effector T cells contributes to the pathogenesis of HAM/TSP. 5) An understanding of the role of regulatory T cells in HAM/TSP had led to the analysis of this T cell subset in patients with MS. Using assays and approaches developed in the HTLV-I - HAM/TSP system, we cannot demonstrate an alteration in the T regulatory cells (either in number or in function) in the peripheral blood of MS patients compared to healthy controls. However, in a clinical trial with humanized anti-IL2 receptor (daclizumab) in MS, we have observed a significant reduction in this important regulatory population in all patients treated. In addition, a surprising number of daclizumab-treated MS patients have manifested skins lesions and attempts are currently underway to determine if this clinical observation is related to the disregulation of T regulatory cells. 6) Using a proteomic approach to generate disease specific protein profiles (Surface Enhanced Laser Desorption Ionization Time-Of-Flight Mass Spectroscopy (SELDI)) we have defined phenotypic signatures within HTLV-1-infected sera that can discriminate HTLV-I infected patients with neurologic disease from patients with HTLV-I associated malignancies. We have extended this work from HAM/TSP to MS and have begun an extensive proteomic profile analysis of sera from patients with multiple sclerosis and controls including healthy individuals and patients with other inflammatory and neurologic disease. Discrete protein profiles have been obtained that distinguish these groups. Bioinformatical and proteomic analytical approaches are being used to determine if signatures can be defined that discriminate severity and type of disease. 7) We continue to extend our work on the detection of the human herpesvirus (HHV-6) from brain resections of patients with mesial temporal lobe epilepsy and patients with neurologic complications following allogeneic bone marrow transplants. We have isolated this virus from explanted primary astrocyte cell cultures and have shown a dysregulation of glutamate uptake in HHV-6 infected astrocyte cell lines. As there are a number of antiviral HHV-6 compounds currently available we have shown that these drugs have different sensitivities in HHV-6 infected glial cells compared to virus-infected lymphocytes. These data suggest that different treatment strategies should be considered when trying to clear this virus from the periphery or the CNS. We have utilized quantitative real-time PCR for the detection of HHV-6 sequences and developed a novel electrochemiluminescent ELISA method for the quantitative detection of antibodies to HHV-6 IgG. We have screened large panels of sera from patients with MS, encephalitis and controls. Preliminary results suggest significantly more HHV-6 DNA in patients with encephalitis of unknown origin than previously reported. In addition, we tested the hypothesis that HHV-6 may be reactivated in MS patients undergoing aggressive immunomodulatory therapy. In particular, we have observed increase virus in CSF of MS patients who have received natalizumab (Tysabri) therapy compared to untreated MS patients. We hypothesize that the reactivation of this virus may lead to transactivation of the JC virus that has been linked to a rare, fatal outcome natalizumab treatment. Collectively, these results continue to define the role of human viruses that are associated with chronic progressive neurologic disease.

人类T淋巴病毒I型（HTLV-I）是一种慢性进行性骨髓病的病因，称为HTLV-I相关的骨髓病/热带痉挛性拼发（HAM/TSP），一种疾病，临床上与多发性孢子菌病的慢性渐进形式相似（MS）。对具有已知病毒病因（HTLV-I和HAM/TSP）神经系统疾病的发病机理的理解将有助于定义MS中类似的发病机理，MS是一种未知病因的疾病，其中怀疑病毒被怀疑起着作用。此外，其他病毒也与MS有关，最近特别关注了无处不在的人疱疹病毒（HHV-6）。对MS中该药物的调查研究导致对其他疾病的研究，这些疾病也可能与病毒有关，包括癫痫，神经系统并发症后同种异体骨髓移植和脑炎。这些研究的主要发现是：1）在HAM/TSP患者的外周血和CSF中，HTLV-I特异性CD8+细胞的增加升高，并且与HTLV-I前病毒DNA和RNA的量直接成正比。这些抗原特异性T细胞被认为是免疫发病的，可能直接参与中枢神经系统中的病毒宿主相互作用。 2）已经证明了对HAM/TSP的脊髓部分中这些抗原特异性CD8+ T细胞以及HTLV-1感染的CD4+细胞的直接观察，并支持这些疾病毒性效应细胞在这种疾病的发病机理中的作用。 3）作为另一个细胞毒性标记，我们证明了自发性脱粒和IFN-gamma产生（由CD107+ IFN-GAMMA+细胞定义）与HTLV-i-I-IN的患者的CD14+巨噬细胞中的前病毒DNA负荷相关，并增强了CD14+ TSP患者在CD14+ THEN+ TSP中的CD8+ TSSCRECT中的IL-15表达。 HTLV-1感染的巨噬细胞的观察是新颖的，这比以前报道的那样对该病毒更广泛。还研究了HTLV-I对CD14+巨噬细胞的影响。 3）CD4+ CD25+调节性T细胞在维持免疫学自我耐受和预防自身免疫性疾病方面很重要。我们已经证明，在HTLV-I感染HAM/TSP患者的CD4+ CD25+ T细胞中，叉子转录因子Foxp3（调节性T细胞的特定标记）的表达低于健康个体的表达。我们已经显示出HAM/TSP患者中FOX-P3阳性T调节细胞的蛋白质表达降低。我们还证明，HTLV-I税对FOXP3表达具有直接的抑制作用，并抑制了这些细胞的调节功能。这些结果表明，通过CD4+ CD25+调节性T细胞的失调，CD4+ CD25+ T细胞的直接人逆转录病毒感染可能与HTLV-I相关神经疾病的发病机理有关。 4）我们还证明，HTLV-I税收基因通过阻断TGF-beta信号传导的机制抑制了FOXP3的表达。 由于已显示TGF-B信号传导对于FOXP3表达和Treg功能至关重要，因此我们还表明，在HAM/TSP患者中，该信号通路失调。我们发现，与正常供体相比，HAM/TSP患者的SMAD7（TGF-β诱导基因）和CD4+ T细胞中CD4+ T细胞中TGF-β受体II（TGF-BRII）的水平显着降低。 此外，TGF-BRII的表达与前病毒载荷成反比。 重要的是，与正常供体相比，来自HAM/TSP患者的CD4+CD25+和CD4+CD25-T细胞表现出降低的TGF-BRII表达，这与体外功能性缺陷有关，包括抑制treg抑制器功能和效应T细胞从TREG介导的对照中逃脱。该证据表明，病毒诱导的影响调节和效应T细胞的免疫耐受性崩溃有助于HAM/TSP的发病机理。 5）对调节性T细胞在HAM/TSP中的作用的理解已导致对MS患者的T细胞子群的分析。使用HTLV -I -HAM/TSP系统中开发的测定和方法，与健康对照组相比，MS患者的周围血液中T调节细胞（数量或功能的数量或功能）的改变。但是，在MS中使用人源化抗IL2受体（Daclizumab）的临床试验中，我们观察到所有接受治疗的患者的重要调节种群的显着降低。此外，目前正在进行了令人惊讶的Daclizumab治疗的MS患者，目前正在进行皮肤病变和尝试，以确定该临床观察是否与疏离T调节细胞有关。 6）使用蛋白质组学方法产生特异性蛋白质谱（表面增强的激光解吸电离飞行时间质谱（SELDI））我们已经在HTLV-1感染的血清内定义了表型特征，可以区分HTLV-I感染HTLV-I相关患者的HTLV-I感染HTLV-I感染的患者。我们已经将这项工作从HAM/TSP扩展到MS，并开始对患有多发性硬化症患者的血清和包括健康个体以及其他炎症性和神经系统疾病的患者进行广泛的蛋白质组学分析。已经获得了区分这些组的离散蛋白质谱。使用生物信息组和蛋白质组学分析方法来确定是否可以定义签名，以区分严重性和类型的疾病。 7）我们继续扩展从检测到颞颞叶癫痫患者的大脑切除术和同种异体骨髓移植后神经系统并发症的患者的大脑切除术中的人疱疹病毒（HHV-6）的工作。我们已经从植入的原发性星形胶质细胞培养物中分离出该病毒，并显示出HHV-6感染的星形胶质细胞细胞系中谷氨酸摄取的失调。由于目前有许多抗病毒HHV-6化合物，因此与感染病毒感染的淋巴细胞相比，这些药物对HHV-6感染的神经胶质细胞具有不同的敏感性。这些数据表明，在试图从外围或中枢神经系统中清除该病毒时，应考虑不同的治疗策略。 我们利用定量实时PCR来检测HHV-6序列，并开发了一种新型的电化学ELISA方法来定量检测到HHV-6 IgG的抗体。我们已经筛选了来自MS，脑炎和对照患者的大量血清。 初步结果表明，起源脑炎患者的HHV-6 DNA明显多于先前报道的。 此外，我们测试了接受侵略性免疫调节治疗的MS患者可以重新激活HHV-6的假设。 特别是，我们观察到与未经治疗的MS患者相比，接受Natalizumab（Tysabri）治疗的MS患者的病毒增加了。 我们假设该病毒的重新激活可能导致与罕见的致命结果纳他珠单抗治疗有关的JC病毒的反式激活。 总的来说，这些结果继续定义与慢性进行性神经系统疾病相关的人类病毒的作用。