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: 10708597
• 负责人: Steven Jacobson
• 金额: $ 89.29万
• 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/10708597
• 关键词: 2019-nCoV; Affect; Allogeneic Bone Marrow Transplantation; Alzheimer' s Disease; Alzheimer' s disease patient; Amyloid beta-Protein; Anti-Retroviral Agents; Antibody Specificity; Area; Astrocytoma; Atrophic; Autopsy; B-Cell Antigen Receptor; B-Lymphocytes; Bioinformatics; Biological Assay; Blood; Bone Marrow Transplantation; Brain; CD19 gene; CD44 gene; CD8-Positive T-Lymphocytes; CD8B1 gene; COVID-19 patient; Cell Line; Cells; Central Nervous System Diseases; Cerebrospinal Fluid; Chromosomes; Chronic; Clinical; Clonal Expansion; Coupled; Cytomegalovirus; DNA; Data; Detection; Development; Disease; Disease Progression; Encephalitis; Epilepsy; Etiology; Event; Excision; Frequencies; Genomics; HHV-6B; HIV; Herpesviridae; Heterogeneity; Human; Human Herpesvirus 4; Human Herpesvirus 6; Human T-lymphotropic virus 1; Human T-lymphotropic virus 2; IL2RA gene; Imaging Techniques; Immune response; Immunophenotyping; In Situ Hybridization; In Vitro; Individual; Infection; Inherited; JC Virus; Laboratories; Lymphocyte; Magnetic Resonance Imaging; Mediating; Methodology; Methods; Microbe; Microfluidics; Multiple Sclerosis; Neurologic; Neurologic Symptoms; Pathogenesis; Pathogenicity; Pathologic; Pathology; Patients; Pattern; Peptides; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Physiologic pulse; Population; Process; Progressive Multifocal Leukoencephalopathy; RNA; Regulatory T-Lymphocyte; Relapsing-Remitting Multiple Sclerosis; Reproducibility; Role; Sampling; Senile Plaques; Spinal Cord; Spinal Cord Diseases; Stable Disease; Surface; System; T cell response; T-Lymphocyte; T-cell receptor repertoire; TNFRSF5 gene; Technology; Temporal Lobe Epilepsy; Testing; Time; Transplant Recipients; Tropical Spastic Paraparesis; Vesicle; Viral; Viral Proteins; Virus; Virus Diseases; Virus Replication; Work; analysis pipeline; antimicrobial peptide; base; cadmium ion; chronic neurologic disease; cohort; cytotoxic; cytotoxic CD8 T cells; detection assay; digital; enzyme linked immunospot assay; exosome; extracellular; extracellular vesicles; healthy volunteer; inflammatory milieu; interest; mind control; multiple sclerosis patient; nervous system disorder; neurotropic virus; novel; particle; pathogen; peripheral blood; post-COVID-19; potential biomarker; single-cell RNA sequencing; small molecule inhibitor; transcriptomics; viral DNA; viral RNA; viral detection

Major findings from our work have focused on the role of immunopathogenic HTLV-I specific CD8+ cells in the pathology of patients with HAM/TSP. We have shown that the frequency of these cells are elevated in the peripheral blood and even higher in the CSF of HAM/TSP and are directly proportional to the amount of HTLV-I proviral DNA and RNA. Based on the development of novel MRI imaging techniques that can quantify spinal cord atrophy in patients with chronic myelopathies, we have correlated the levels of cytotoxic CD8+ T cells with the extent of spinal cord atrophy in HAM/TSP patients. We have extended the cross-sectional spinal cord analyses to other neurological diseases including multiple sclerosis (MS). We are demonstrating a heterogeneity of atrophic patterns in relapsing-remitting MS. We have developed ELISPOT assays for the detection of virus-specific immune responses to a variety of neurotropic viruses including EBV, CMV and HHV6. We have shown that immunodominant peptides of EBV induce T cell responses to unique regions of EBV in patients with active MS in comparison to healthy controls or MS patients with stable disease. In addition, we have shown that purified CD19+ B cells from active MS patients have a higher frequency of EBV detection than in stable MS patients or control cohorts. These results support a role for EBV as a potential trigger in this disease. We are currently using small molecule inhibitors to interfere with EBV replication in B cells and PBMC of patients with MS. Our analysis of T cell receptor repertoires in the peripheral blood and CSF of HAM/TSP and MS patients and have demonstrated T cell clonal expansions particularly in the CSF of MS patients. We have also begun to explore both the T and B cell receptor repertoires from single cells in the CSF of these patients and have successfully expressed paired Vh and Vl chains. The specificity of these antibodies is being investigated. In addition, we have begun single cells RNAseq analysis of PBC and CSF to define the TCR and BCR repertoire of patients with chronic neurologic diseases including MS, HAM/TSP and long-term post COVID-19 patients with neurologic sequelae. We have examined the role of exosomes and extracellular vesicles (EVs) from blood and CSF of patients with known virus-associated neurologic disease including HAM/TSP (HTLV-I) and PML (JC virus). Using multi-parameter immunoflow technology, we have previously characterized these vesicles and determined their cargo for the presence of viral proteins and viral RNAs. Recently, we have analyzed the EVs from the CSF of healthy volunteers and patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies including MS, HAM/TSP, HTLV-1-infected asymptomatic carriers, and other neurological diseases (ONDs), to investigate the surface repertoires of CSF EVs during disease. Significant increases in CD8+ and CD2+ EVs were found in HAM/TSP patient CSF samples compared to other clinical groups, consistent with the immunopathologically-mediated disease associated with CD8+ cells in the CNS of HAM/TSP patients. Furthermore, CD8+, CD2+, CD44+, and CD40+ EVs were significantly increased in the CSF from patients with viral infections compared to those without. These data suggest that CD8+ and CD2+ CSF EVs may be important as: 1) potential biomarkers for viral-mediated neurological diseases, and 2) as possible meditators of areas of the disease process in infected individuals. Additionally, we have made significant advances in the analysis pipeline for analyzing the concentration of EVs and extracellular particles in biofluids including CSF through microfluidic resistive pulse sensing (MRPS). We can now analyze and compare the concentrations of EVs between disease groups with great reproducibility and accuracy. The purpose of these ongoing studies is to determine if at times when no virus can be detected, EVs particularly from the CSF will carry a viral signature or pattern that can be discerned. We are continuing to assess virus-specific immune responses from MS patients and controls using virus-peptide pools and can demonstrate EBV-specific CD4+ and CD8+ T cells that can be detected by HLA-restricted tetramers. In addition, we are using our newly developed digital based PCR methodology (ddPCR) for the detection of human viruses. We have characterized PCR primer and pair probes for the detection of HTLV-I, HTLV-II, EBV, JCV, CMV and HHV6 A and HHV6B. We have multiplexed this system in which we can now simultaneously detect and quantify herepesvirus sequences in human material. We have also developed this assay for the detection of SarsCOV2 and will explore the extent and magnitude of this virus in recovering COVID19 patients with neurologic signs and symptoms. 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, patients with neurologic complications following allogeneic bone marrow transplants, and patients with astrocytomas. We have been exploring the clinical consequences of inherited chromosomally integrated HHV-6 (iciHHV-6) which affects approximately 1% of the human population. ciHHV6 integrates into host chromosomes at one copy of HHV-6 DNA per cell while it remains unclear if HHV-6 reactivates from this integrated state. T cell lines have been generated from iciHHV-6B PBMC and will be tested for reactivation (by a variety of compounds know to stimulate HHV6) by viral expression using PCR assays targeting HHV-6B DNA or RNA, and RNAscope for in situ hybridization. We can induce iciHHV-6 lymphocytes to express HHV-6 RNA in the absence of an increasing viral DNA load. Recently, there has been a re-kindled interest in the infectious-trigger hypothesis of Alzheimers disease (AD) based on bioinformatic analysis of large cohorts of AD autopsy brains compared to controls in which HHV6 was suggested to be associated with AD. This coupled with the observation that extracellular beta-amyloid plaques (a pathologic hallmark of AD) may function as an innate antimicrobial peptide that is released to entrap pathogens and protect the brain from infection, has suggested that Abeta can be nucleated and seeded by microbes in vitro, causing aggregation and subsequent plaque formation. We have performed a bioinformatics and PCR based analysis, on large cohort of AD and control brain samples. Although we were not able to amplify HHV-6 or other candidate viruses from AD patient samples, this did not preclude a possible association with an early triggering event, rather than disease progression, or these agents may be present at copy numbers below the limit of laboratory detection.

我们工作的主要发现集中在免疫致病性HTLV-I特异性CD8+细胞在HAM/TSP患者病理学中的作用。 我们已经表明，这些细胞的频率在外周血中升高，在HAM/TSP的CSF中甚至更高，并且与HTLV-I前病毒DNA和RNA的量直接成正比。 基于可以量化慢性骨髓病患者脊髓萎缩的新型MRI成像技术的发展，我们将细胞毒性CD8+ T细胞的水平与HAM/TSP患者的脊髓萎缩程度相关联。 我们已经将横断面脊髓分析扩展到其他神经系统疾病，包括多发性硬化症（MS）。 我们正在展示复发型MS中萎缩模式的异质性。 我们开发了ELISPOT分析，用于检测对包括EBV，CMV和HHV6在内的多种神经机关病毒的病毒特异性免疫反应。我们已经表明，与健康对照组或患有稳定疾病的MS患者相比，EBV的免疫主导肽对活性MS患者的独特区域EBV诱导T细胞反应。 此外，我们已经表明，与稳定的MS患者或对照组相比，活跃MS患者的纯化CD19+ B细胞的EBV检测频率更高。 这些结果支持EBV作为该疾病的潜在触发作用。 我们目前正在使用小分子抑制剂来干扰MS患者的B细胞和PBMC中的EBV复制。 我们对HAM/TSP和MS患者的外周血和CSF中T细胞受体曲目的分析，并证明了T细胞克隆扩张，特别是在MS患者的CSF中。 我们还开始探索这些患者CSF中单个细胞的T和B细胞受体库，并成功表达了配对的VH和VL链。 这些抗体的特异性正在研究。此外，我们已经开始对PBC和CSF进行单细胞RNASEQ分析，以定义患有慢性神经系统疾病患者的TCR和BCR曲目，包括MS，HAM/TSP和Covid后19例神经系统后遗症患者。 我们已经检查了来自血液和CSF的外泌体和细胞外囊泡（EV）的作用，包括已知病毒相关的神经系统疾病，包括HAM/TSP（HTLV-I）和PML（JC病毒）。使用多参数免疫流技术，我们先前已经表征了这些囊泡，并确定了它们的货物，以便存在病毒蛋白和病毒RNA。 Recently, we have analyzed the EVs from the CSF of healthy volunteers and patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies including MS, HAM/TSP, HTLV-1-infected asymptomatic carriers, and other neurological diseases (ONDs), to investigate the surface repertoires of CSF EVs during disease.与其他临床组相比，在HAM/TSP患者CSF样品中发现CD8+和CD2+ EV的显着增加，这与HAM/TSP患者中枢神经系统中与CD8+细胞有关的免疫病理学介导的疾病一致。此外，与没有病毒感染的患者相比，CSF的CD8+，CD2+，CD44+和CD40+EV显着增加。这些数据表明，CD8+和CD2+ CSF EV可能很重要，因为：1）病毒介导的神经系统疾病的潜在生物标志物，以及2）在受感染个体中疾病过程的冥想者可能是冥想者。此外，我们在分析管道中取得了重大进步，用于分析包括通过微流体电阻式脉冲传感（MRP）在内的生物流体中电动汽车和细胞外颗粒的浓度。现在，我们可以分析和比较具有很高可重现性和准确性的疾病组之间电动汽车的浓度。这些正在进行的研究的目的是确定是否有时无法检测到病毒，尤其是CSF的EV会带有可以识别的病毒特征或模式。 我们将继续评估使用病毒肽池的MS患者和对照组的病毒特异性免疫反应，并可以证明EBV特异性的CD4+和CD8+ T细胞可以通过HLA限制的四聚体检测到。此外，我们正在使用新开发的基于数字的PCR方法（DDPCR）来检测人类病毒。我们已经表征了PCR引物和配对探针，以检测HTLV-I，HTLV-II，EBV，JCV，CMV和HHV6 A和HHV6B。我们已经多样地进行了多元化的系统，现在我们可以同时检测和量化人类材料中的佩皮病毒序列。我们还开发了用于检测SARSCOV2的测定法，并将探讨该病毒在恢复具有神经系统症状和症状的COVID19患者方面的程度和大小。 我们继续扩展从检测到人疱疹病毒（HHV-6）的研究中，从中颞叶癫痫患者的大脑切除术，同种异体骨髓移植后神经系统并发症的患者以及星形胶质细胞瘤患者。我们一直在探索遗传性染色体综合HHV-6（ICIHHV-6）的临床后果，这影响了大约1％的人口。 CIHHV6在每个细胞的HHV-6 DNA副本上集成到宿主染色体中，而HHV-6是否从该集成状态重新激活。 T细胞系是由ICIHHV-6B PBMC产生的，将通过使用针对HHV-6B DNA或RNA的PCR测定法进行重新激活（通过多种化合物来刺激HHV6）进行重新激活（通过多种化合物），并进行rnascope，并进行rnascope以进行原位杂交。 在没有病毒DNA载荷增加的情况下，我们可以诱导ICIHHV-6淋巴细胞表达HHV-6 RNA。最近，与建议HHV6与AD相关的对照组相比，基于对大型AD尸检大脑的生物信息学分析，基于对大量AD尸检大脑的生物信息学分析，对阿尔茨海默氏病（AD）的感染触发假设产生了重新引起的兴趣。 这与观察到的观察到，细胞外β-淀粉样木质斑（AD的病理标志）可能是一种先天抗菌肽，该抗菌肽被释放以诱发病原体并保护大脑免受感染的侵害，这表明Abeta可以由Microbes在Vitro，Vitro，Vitro，Controce-Contrication Plaque-Sequecration plaque Plaque plaque plaque plaque plaque plaque plaque plaque Plaque plaque plaque。 我们对大量AD和对照脑样品进行了生物信息学和基于PCR的分析。 尽管我们无法从AD患者样本中扩增HHV-6或其他候选病毒，但这并不排除可能与早期触发事件而不是疾病进展的可能关联，或者这些药物可能以低于实验室检测极限的拷贝数出现。