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

在2020财年，我们加快了使用体外方法的研究计划。一个关键的体外项目是使用人全血培养系统筛选一些紫锥菊提取物和来自这些提取物的成分。使用他的培养系统，我们研究了在紫锥菊化合物存在下用各种免疫激活剂体外刺激后人白色血细胞的细胞因子释放。这些体外研究中产生的数据有助于选择紫锥菊提取物用于紫锥菊的体内免疫毒性评估。正在进行的项目正在使用体外人全血培养系统调查个体间易感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试验系统对研究PAC化合物免疫毒性的适用性。这些数据将参考先前在PAC-MAP混合物研究中收集的小鼠体内数据，并用于锚人体相关性并加强可使用体外系统生成的数据。BRT目前正在开发用于该培养系统的其他体外工具，这将有助于询问体液抗体介导的免疫和T细胞驱动的细胞介导的免疫。这种体外工具箱对于免疫毒性测试的未来将是非常宝贵的，并将使NTP处于该领域测试的最前沿。最近，潜在致敏物的体外危害识别技术引起了NTP的极大兴趣。 在2019财年，BRT完成了一系列检测的方法转移和优化，以识别可能导致致敏和超敏反应的化学品。选择用于NTP免疫毒理学合同的试验包括以下内容：1）DPRA -直接肽反应性试验; 2）角蛋白-角质形成细胞活化; 3）h-CLAT -人细胞系活化试验，树突状细胞。这些试验现在将作为NTP体外试验组合中的主要试验，用于评价化学品引起致敏的可能性。NTP与EPA，CSPC和其他联邦机构合作，编制了一份186种化学品的清单，这些化学品是体外测试的候选者。NTP确定了另外14种化学品，另外27种农药由一个行业伙伴提供。对这227种化学品的评估已于2020财年完成。该项目已产生6份报告草稿，其中3份现已结束。另有3份报告正在定稿过程中。 新型体外检测策略的开发和评价是免疫毒理学领域的一项重大研究举措。在2020财年，我们与行业合作伙伴签订了一项合作研究协议，以评估GARD检测试剂盒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.