STUDIES TO EVALUATE THE POTENTIAL FOR ENVIRONMENTAL&THERAPEUTIC AGENTS TO INDUCE IMMUNOTOXICITY - In vitro

在2020财年，我们加快了体外方法的研究计划。一个关键的体外项目是筛选一些紫锥菊提取物和从这些提取物中提取的成分使用人全血培养系统。利用他的培养系统，我们检测了在紫锥菊化合物存在的情况下，在体外用各种免疫激活剂刺激人白细胞后细胞因子的释放。在这些体外研究中产生的数据有助于选择一种紫锥菊提取物用于紫锥菊的体内免疫毒性评估。正在进行的项目是使用体外人全血培养系统调查个体间易感性因素和环境风险因素。终点包括淋巴细胞毒性、细胞因子释放和自然杀伤细胞活性。第二个项目将使用相同的培养系统和终点来研究测试系统在研究PAC化合物免疫毒性方面的适用性。这些数据将与之前在PAC-MAP混合物研究中收集的小鼠体内数据进行参考，并用于确定人类相关性并加强使用体外系统可以产生的数据。BRT目前正在为该培养系统开发额外的体外工具，以促进对体液抗体介导的免疫和t细胞驱动的细胞介导的免疫的询问。这个体外工具箱对于免疫毒性测试的未来将是无价的，并将国家毒理学控制项目置于该领域测试的前沿。最近，潜在致敏物的体外危险识别技术引起了国家毒理学规划的极大兴趣。在2019财年，BRT完成了一系列测试的方法转移和优化，用于识别可能导致致敏和超敏的化学品。NTP免疫毒理学合同选择的检测包括以下内容：1)DPRA -直接肽反应性测定；2)角质蛋白-角质细胞活化；3) h-CLAT -人细胞系活化试验，树突状细胞。这些测定法现在将作为国家毒理学规划体外测试系统的主要测定法，用于评估化学物质引起致敏的可能性。国家毒理学规划与EPA、CSPC和其他联邦机构合作，编制了一份186种可供体外测试的候选化学品清单。国家毒理学规划确定了另外14种化学品，一个行业伙伴提供了另外27种农药。对这227种化学品的评估已于2020财年完成。该项目产生了6份报告草案，其中3份现已完成。另有3份报告正在最后定稿过程中。开发和评估新的体外检测策略是免疫毒理学领域的一项重要研究。在2020财年，我们与一家行业合作伙伴达成了一项合作研究协议，以评估GARD检测作为评估超敏反应的额外体外工具。

In FY 20 we have accelerated the research program using in vitro approaches. A key in vitro project was the screening of a number of echinacea extracts and constituents derived from these extracts using a human whole blood culture system. Using his culture system, we examined cytokine release from human white blood cells following stimulation in vitro with various immune activators in the presence of the echinacea compounds. The data generated in these in vitro studies were instrumental in the selection of an echinacea extract to be used an in vivo immunotoxicity assessment of echinacea purpurea. Ongoing projects are investigating interindividual susceptibility factors and environmental risk factors using an in vitro human whole blood culture system. Endpoints include lymphocytotoxicity, cytokine release, and Natural Killer cell activity. The second project will use the same culture system and endpoints to investigate the applicability of the test system to investigating the immunotoxicity of PAC compounds. These data will be referenced against mouse in vivo data previously collected in the PAC-MAP mixtures studies and serve to anchor human relevance and strengthen the data that can be generated using the in vitro system. BRT is currently working on development of additional in vitro tools for this culture system that will facilitate interrogation of humoral antibody mediated immunity and T-cell driven cell-mediated immunity. This in vitro toolbox will be invaluable for the future of immunotoxicity testing and will position NTP at the cutting edge of testing in this field. Recently, in vitro techniques for hazard identification for potential sensitizers have been of great interest to the NTP. In FY19, BRT completed method transfer and optimization for a battery of tests for identification of chemicals with the potential to cause sensitization and hypersensitivity. The assays selected for the NTP Immunotoxicology contract included the following: 1) DPRA – Direct Peptide Reactivity Assay; 2) Keratinosens – keratinocyte activation; 3) h-CLAT – Human cell line activation assay, dendritic cells. These assays will now serve as the main assays in the NTP in vitro testing battery that will be used to evaluate the potential for chemicals to cause sensitization. The NTP, in collaboration with the EPA, CSPC, and other federal agencies, has compiled a list of 186 chemicals that are candidates for in vitro testing. 14 additional chemicals were identified by the NTP and an additional 27 pesticides were provided by an industry partner. Assessment of these 227 chemicals was completed in FY20. This project has led to the generation of 6 draft reports, 3 of which have now been closed. An additional 3 reports are in the process of being finalized. Development and evaluation of novel in vitro testing strategies is a major research initiative in the field of immunotoxicology. In FY20 we entered into a collaborative research agreement with an industry partner to evaluate the GARD assay as an additional in vitro tool for the assessment of hypersensitivity.