Improving The Sensitivity And Predictability Of Testing

作为NIEHS艾滋病工作的一部分，NIEHS和FDA之间的机构间协议正在进行的研究正在确定免疫细胞表型减少与感染或肿瘤易感性之间的关系。在FDA的James Weaver博士的实验室中，使用抗CD4和CD8的单抗(Moabs)在B6C3F1小鼠中建立这些细胞亚群耗尽的滴定曲线的研究已经完成并发表。在NIEHS，我们正在研究特定宿主耐药模型中细胞耗尽的影响。我们目前正在评估在SA1肿瘤模型中CD4细胞耗尽的影响。已有文献报道，这种肿瘤的清除高度依赖于细胞介导的免疫反应，尽管CD4和CD8T细胞的相对贡献仍有待解决。我们还评估了特定免疫细胞群耗尽和COX缺陷小鼠对流感的反应。COX-1基因缺失和野生型小鼠在流感攻击后第1天体重和体温发生了显著变化，但小鼠似乎恢复并清除了感染。相反，COX-2基因缺失的小鼠在早期表现出体温降低和体重减轻，但在第3天出现加速反应。50%的COX-2基因缺失小鼠在第5-6天死亡，而COX-1基因缺失或野生型小鼠没有死亡。COX-2基因缺陷小鼠肺泡灌洗液中促炎症细胞因子TNFα、IL-1β和抗病毒细胞因子IFN-γ水平显著降低，中性粒细胞和巨噬细胞向呼吸道的募集明显减弱。我们目前正在评估这些小鼠组织中的相对病毒负荷。 过去15年使用标准化测试小组收集的实验动物数据提供了一个数据库，从该数据库评估了免疫毒性筛选化学品常用的各种测试的敏感性和可预测性。这些结果已被用作免疫毒性风险评估的指南，并已成为一些监管活动的基础。扩展组织病理学主要研究淋巴器官的结构和构筑变化，作为免疫毒性评估的主要筛选试验，已经引起了相当大的兴趣。为了确定这种方法作为独立筛查的实用性，启动了一项使用国家毒理学计划免疫毒理学测试计划数据的验证工作。这项研究使用了十种测试化学物质和阴性和阳性对照的数据集，解决了扩展组织病理学的实验室间重复性。我们检查了具有不同背景的病理学家在评估免疫组织中的损伤时的一致性，以及个体和联合组织病理学终点检测化学效应和剂量反应的敏感性。病理学家之间在胸腺的符合率最高，特别是胸腺皮质细胞的符合率，而在脾和淋巴结检查的所有间隔中符合率较低。此外，分析表明，准确识别淋巴器官组织病理学变化的能力直接取决于个人在免疫组织学方面的经验/培训以及特定病变的明显严重程度。

Studies being conducted as part of an interagency agreement between NIEHS and FDA as part of the NIEHS AIDS effort are determining the relationship between decrements in immune cell phenotypes and susceptibility to infection or tumors. Studies conducted in the laboratory of Dr. James Weaver at FDA, using monoclonal antibodies (Moabs) against CD4 and CD8, to establish titration curves for the depletion of these cell subpopulations in B6C3F1 mice have been completed and published. At NIEHS, we are examining the effects of cell depletion in specific host resistance models. We are currently evaluating the effects of CD4+ cell depletion in the SA1 tumor model. It has been reported in the literature that clearance of this tumor is highly dependent on cell-mediated immune responses, although the relative contributions of CD4+ and CD8+ T cells remain to be addressed. We have also evaluated responses to influenza following depletion of specific immune cell populations and in COX deficient mice. Significant changes in body weight and temperature were observed in COX-1 null and wild type mice 1 day following influenza challenge, however the mice appeared to recover and clear the infection. In contrast, COX-2 null mice showed a lack of hypothermia and weight loss at early time points, but an accelerated response on day 3. Fifty percent of COX-2 null mice died between days 5 and 6 whereas there was no mortality in COX-1 null or wild type mice. Levels of the proinflammatory cytokines TNFalpha and IL-1beta and the anti-viral cytokine IFNgamma were dramatically reduced in bronchioalveolar lavage fluid from COX-2 deficient mice and the recruitment of neutrophils and macrophages to the airways was markedly attenuated these animals. We are currently assessing the relative viral burden in the tissues of these mice. Experimental animal data collected over the past 15 years using standardized testing panels has provided a database from which the sensitivity and predictability of a variety of tests commonly used for the screening of chemicals for immunotoxicity has been evaluated. These results have been used as guidelines for risk assessment in immunotoxicity and have been the basis for a number of regulatory activities. There has been considerable interest in the use of expanded histopathology, which focuses on examining the structural and architectural changes in lymphoid organ, as a primary screening test for immunotoxicity assessment. To determine the utility of this approach as a stand alone screen, a validation effort using data from the National Toxicology Program's immunotoxicology testing program was initiated. This study addresses the interlaboratory reproducibility of extended histopathology using a dataset of ten test chemicals and both negative and positive controls. We examined the consistency between pathologists with varied background in evaluating lesions in immune tissues and the sensitivity of the individual and combined histopathological endpoints to detect chemical effects and dose response. Agreement between pathologists was highest in the thymus, in particular with thymic cortical cellularity, and lower within all of the compartments examined in the spleen and lymph nodes. In addition, the analyses indicated that the ability to accurately identify histopathological change in lymphoid organs is dependent directly upon the experience/training that the individual possesses in immunohistology and the apparent severity of the specific lesion.