Human Specimen Collection to Support Basic and Clinical Research

支持基础和临床研究的人体样本采集

• 批准号: 10929189
• 负责人: Arun Shet
• 金额: $ 22.89万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/10929189
• 关键词: Acute; Address; Anemia; Antibodies; Anticoagulation; Antisickling Agents; Autologous; Basic Science; Biochemical; Biological Assay; Biological Markers; Biology; Biotin; Blood; Blood Platelets; Blood Vessels; Blood specimen; Bone Marrow; CD34 gene; Cell Adhesion Molecules; Cell Volumes; Cells; Clinical Research; Clinical Trials; Coagulation Process; Collaborations; Collection; DNA; Data; Density Gradient Centrifugation; Development; Disease; Engraftment; Equilibrium; Erythrocytes; Ethnic Origin; Fiber; Flow Cytometry; Functional disorder; Gene Expression Profiling; Gene Transfer; Genetic; Half-Life; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cell heterogeneity; Hematopoietic stem cells; Hemoglobin; Human; Image; In Vitro; Individual; Inflammation; Intramural Research Program; Isomerase; Kinetics; Label; Laboratories; Laboratory Chemicals; Laboratory Research; Libraries; Longevity; Marrow; Measurement; Measures; Methodology; Methods; Mitochondrial DNA; Modification; Molecular; National Heart, Lung, and Blood Institute; National Human Genome Research Institute; Neutrophil Activation; Oxygen; PF4 Gene; Pathogenesis; Pathologic Processes; Patients; Physics; Plasma; Plasma Proteins; Platelet aggregation; Polymerase Chain Reaction; Preparation; Protein Disulfide Isomerase; Protocols documentation; Research; Research Support; Reticulocytes; Saliva; Sampling; Serum; Sickle Cell; Sickle Cell Anemia; Sickle Cell Trait; Sickle Hemoglobin; Specimen; Standardization; Testing; Thermodynamics; Thrombospondins; Urine; Variant; Vascular Diseases; Venous Thrombosis; Whole Blood; assay development; biological development; bisulfite sequencing; cell free DNA; comparison control; cytokine; density; endothelial dysfunction; experimental study; extracellular; extracellular vesicles; genetic approach; genome sequencing; granulocyte; healthy volunteer; in vitro Assay; inter-individual variation; laboratory development; luminescence; neutrophil; phase 2 study; progenitor; protein expression; protein function; research and development; research study; sample collection; sickling; single-cell RNA sequencing; small molecule inhibitor; vaso-occlusive crisis; volunteer; whole genome

The overall scope and purpose of this research protocol is to support assay development and research conducted within the NHLBI and other divisions of the intramural research program, especially the Sickle Cell Branch. Biospecimens, typically include blood samples that are collected from subjects, including those with sickle cell trait and subjects with sickle cell disease. The various methods and assays developed by different collaborators are listed below. The Sickle Cell Branch is focused on developing a greater understanding of acute pathophysiology, genetics and vascular biology in Sickle Cell Disease. The following assays were developed in the sickle cell branch laboratories: 1. Platelet function assays: Platelets isolated from whole blood, to study in vitro platelet aggregation studies. Whole blood was used to study platelet aggregation and ATP luminescence. The protocol has provided samples to standardize the assay using healthy ethnic matched controls. 2. Cell-Free DNA and mitochondrial DNA assays: Optimization of cell-free DNA extraction, quantitation, and library preparation methodology. Whole genome sequencing (WGS) and whole genome bisulfite sequencing (WGBS), and quantitative polymerase chain reaction (qPCR), where the cell-free DNA extracted from healthy volunteers was used as an independent healthy control to compare with sickle cell disease patients who are in steady-state and crisis. 3. Neutrophil Activation and Extracellular Trap Formation: Blood from healthy volunteers and plasma from sickle cell patients were used for in vitro neutrophil functional studies (neutrophil extracellular trap formation). In separate experiments blood from both healthy donors and sickle cells patients are used to isolate low density granulocytes (LDGs) using density gradient centrifugation and flow cytometry. 4. Cell derived extracellular vesicles (EVs) in plasma: Assays to study plasma borne cell-derived EVs obtained from SCD patients and Healthy volunteers are being standardized for two newly established research protocols to study the impact of acute vaso-occlusive crisis and venous thrombosis on plasma EV numbers and coagulation profile. 5. Assays to quantify and study the isomerase function of protein disulfide isomerase (PDI) have been standardized using this protocol. The results of this assay will be used to support research conducted in a phase 2 study of a small molecule inhibitor of plasma PDI. The Cellular and Molecular Therapy Branch is focused on developing genetic strategies aimed at correction of SCD through modification of autologous hematopoietic stem cells (HSCs) from the marrow and on hematopoietic stem cell transplantation as a cure for SCD. The following assays are under development in the Cellular and Molecular Therapy Branch: 1. In vitro modification of autologous hematopoietic stem cells (HSCs) from the marrow in an ongoing clinical trial testing lentiviral gene transfer to HSCs in patients with SCD. Therefore, bone marrow continues to be collected from volunteer patients to optimize cell processing and HSC enrichment. Data are analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating variations in antibody labeling intensity to characterize HSC heterogeneity and progenitor lineage. 2. In other studies, blood is obtained from sickle cell patients and healthy controls to standardize cytokine measurements in SCD patients and healthy controls and compare them with SCD patients who underwent haploidentical hematopoietic stem cell. Measurements include assays for thrombospondin and platelet factor 4 as serum biomarkers of engraftment. 3. Studying the life span of erythrocytes that are modified by either gene transfer or HSC transplantation is critical to understanding the impact of such therapies on anemia in SCD. Assays to study erythrocyte half-life using biotin labeling are currently supported by this protocol. 4. In collaboration with NHGRI, an assay to isolate reticulocytes from anticoagulated whole blood obtained from SCD patients and standardize single cell RNA sequencing of reticulocytes is being developed. This methodology has been successfully completed. The Laboratory of Chemical Physics is concerned with studying the thermodynamics, kinetics, and mechanism of fiber formation of hemoglobin S, and its relation to the pathophysiology and therapy of sickle cell disease. This protocol supports laboratory research by providing blood from sickle cell patients to develop assays that quantify the rate of sickling and the effects of cell volume, hemoglobin concentration on sickling kinetics. 1. These approaches are being used to test the effects of anti-sickling drugs and to study the effects of human variation on the rate of sickling. Specifically, the effect of anti-sickling drugs is being evaluated in healthy controls and those with sickle cell trait. 2. Studies to standardize assays to measure oxygen equilibrium curves in the presence of anti-sickling agents are also underway including the inter-individual variability of these assays.

本研究方案的总体范围和目的是支持在NHLBI和校内研究项目的其他部门（尤其是镰状细胞分支）内进行的测定开发和研究。生物样本通常包括从受试者收集的血液样品，包括具有镰状细胞性状的受试者和患有镰状细胞病的受试者。下面列出了不同合作者开发的各种方法和测定。 镰状细胞分支的重点是发展急性病理生理学，遗传学和血管生物学在镰状细胞病的更好的理解。镰状细胞分支实验室开发了以下试验： 1.血小板功能测定：从全血中分离血小板，以研究体外血小板聚集研究。全血用于研究血小板聚集和ATP发光。方案提供了样本，以使用健康种族匹配对照品标准化试验。 2.游离DNA和线粒体DNA测定：游离DNA提取、定量和文库制备方法的优化。全基因组测序（WGS）和全基因组亚硫酸氢盐测序（WGBS），以及定量聚合酶链反应（qPCR），其中从健康志愿者中提取的游离DNA被用作独立的健康对照，以与处于稳态和危象的镰状细胞病患者进行比较。 3.中性粒细胞活化和细胞外陷阱形成：将来自健康志愿者的血液和来自镰状细胞患者的血浆用于体外中性粒细胞功能研究（中性粒细胞细胞细胞外陷阱形成）。在单独的实验中，来自健康供体和镰状细胞患者的血液用于使用密度梯度离心和流式细胞术分离低密度粒细胞（LDG）。 4.血浆中细胞衍生的细胞外囊泡（EV）：研究从SCD患者和健康志愿者获得的血浆来源的细胞衍生EV的试验正在标准化，用于两个新建立的研究方案，以研究急性血管闭塞危象和静脉血栓形成对血浆EV数量和凝血特征的影响。 5.已使用该方案标准化了定量和研究蛋白质二硫键异构酶（PDI）异构酶功能的测定。本试验的结果将用于支持在血浆PDI小分子抑制剂的II期研究中进行的研究。 细胞和分子治疗分支专注于开发遗传策略，旨在通过修饰来自骨髓的自体造血干细胞（HSC）来纠正SCD，并将造血干细胞移植作为SCD的治愈方法。细胞和分子治疗分支正在开发以下检测试剂盒： 1.在一项正在进行的临床试验中，对来自骨髓的自体造血干细胞（HSC）进行体外修饰，测试SCD患者中慢病毒基因转移至HSC。因此，继续从志愿者患者收集骨髓以优化细胞处理和HSC富集。通过常规流式细胞术和成像流式细胞术分析数据，后者证实了CD34+选择后的流动数据，并证明了抗体标记强度的变化，以表征HSC异质性和祖细胞谱系。 2.在其他研究中，从镰状细胞患者和健康对照中获得血液，以标准化SCD患者和健康对照中的细胞因子测量，并将其与经历单倍体相合造血干细胞的SCD患者进行比较。测量包括测定血小板反应蛋白和血小板因子4作为植入的血清生物标志物。 3.研究通过基因转移或HSC移植修饰的红细胞的寿命对于理解此类疗法对SCD贫血的影响至关重要。本方案目前支持使用生物素标记研究红细胞半衰期的试验。 4.与NHGRI合作，正在开发一种从SCD患者的抗凝全血中分离网织红细胞并标准化网织红细胞单细胞RNA测序的检测方法。这一方法已成功完成。 化学物理实验室致力于研究血红蛋白S纤维形成的热力学、动力学和机制，及其与镰状细胞病的病理生理学和治疗的关系。该方案通过提供镰状细胞患者的血液来支持实验室研究，以开发定量镰状化速率以及细胞体积、血红蛋白浓度对镰状化动力学的影响的测定。 1.这些方法正被用来测试抗镰状化药物的效果，并研究人类变异对镰状化率的影响。具体来说，抗镰状化药物的效果正在健康对照组和镰状细胞性状的人中进行评估。 2.在抗镰状化剂存在下测量氧平衡曲线的标准化测定法的研究也在进行中，包括这些测定法的个体间变异性。

项目成果

Phenotypic screening of the ReFRAME drug repurposing library to discover new drugs for treating sickle cell disease.

• DOI: 10.1073/pnas.2210779119
• 发表时间: 2022-10-04
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1

Voxelotor treatment of a patient with sickle cell disease and very severe anemia.

Voxelotor 对患有镰状细胞病和非常严重贫血的患者进行治疗。

• DOI: 10.1002/ajh.25389
• 发表时间: 2019
• 期刊: American journal of hematology
• 影响因子: 12.8
• 作者: Shet,ArunS;Mendelsohn,Laurel;Harper,Julia;Ostrowski,David;Henry,EricR;Gwaabe,Eveline;Nichols,Jim;Alayash,AbduI;Eaton,WilliamA;Thein,SweeLay
• 通讯作者: Thein,SweeLay

Pro-inflammatory cytokines associate with NETosis during sickle cell vaso-occlusive crises.

• DOI: 10.1016/j.cyto.2019.154933
• 发表时间: 2020-03
• 期刊: Cytokine
• 影响因子: 3.8
• 作者: Barbu EA;Mendelsohn L;Samsel L;Thein SL
• 通讯作者: Thein SL