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。针对CD45R/B220和TCRb抗体的初步研究正在进行中。一种对小鼠外周血进行连续表型分析的方法已经得到验证，一份报告这些结果的手稿已经提交出版。对于单个时间点和时间进程实验，数据都是可比较的。抗CD8单抗的时间进程实验表明，可接受的耗竭水平在单次耗竭注射后至少延长到24天。CD8细胞的耗尽对CD4或B细胞(CD45R+)水平没有显著影响。抗CD4单抗也可以达到可接受的耗竭水平，然而，需要多次耗尽注射5-10微克的抗体。CD4+细胞耗竭对循环CD8+细胞水平无明显影响。在NIEHS，我们已经开始在为这些研究选择的特定宿主耐药模型中检查细胞耗尽的影响。这些模型包括使用细菌(单核细胞增生性李斯特菌或肺炎链球菌)、细胞内寄生虫(约氏疟原虫)或病毒(流感)以及肿瘤挑战(PYB6或B16F10)的感染。对于每个细胞亚群，将评估三种寄主抗性模型，所选的模型取决于特定的目标细胞。在CD8+细胞耗尽的小鼠身上，已经完成了两次PYB6肿瘤挑战。正如预期的那样，在CD8耗尽后，PYB6肿瘤的频率或潜伏期没有显著差异。一项针对CD4耗尽小鼠的PYB6肿瘤研究正在进行中。确定可能对免疫系统造成损害的化学物质具有相当大的公共卫生意义，因为免疫功能的改变会导致过敏性疾病、自身免疫性或传染性疾病或肿瘤的发生率增加。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。1998年4月，NIEHS举行了一次研讨会，以建立研究设计，以确定扩展组织病理学作为免疫毒性指标的敏感性和可预测性，并与国家毒理学计划-S功能测试电池进行比较。为10种化学物质生成了标准化的幻灯片集，此前已使用功能测试对其免疫毒性进行了评估。对幻灯片的组织学评估已经完成，数据已被纳入数据库。拉塞尔·赫尔姆斯博士和最初风险评估研究的合作者克里斯·波蒂埃博士已经完成了病理评分的数据分析。数据表明，对于大多数结果，病理学家之间有很好的一致性。然而，即使变量具有很好的一致性，也有一些病理学家之间不一致的证据。对每个病理学家的评分进行直接比较表明，即使在一致性很好的情况下，某些人往往更保守，而其他人比其他人更能察觉到细微的变化。其他分析通过调查同一组织类型中所有测量之间的相关性来检查病理学家在单个组织中评级的一致性。当所有病理学家的数据结合在一起时，四个组织中每一个组织的测量结果似乎都与该组织的其他测量结果高度相关。然而，当独立检查每个病理学家的相关性时，只有胸腺评估保持了同样的高度相关性。对于脾、骨髓和淋巴结指标，每个病理学家的结果相关性较差，这引发了人们对病理学家在每种组织中不同指标的评级一致性的担忧。一份报告这些发现的手稿正在准备中。

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. Pilot studies with antibodies to CD45R/B220 and TCRb are in progress. A method for serial phenotypic analysis of mouse peripheral blood has been validated and a manuscript reporting these results has been submitted for publication. Data are comparable for both single time point and time course experiments. Time course experiments with the anti-CD8 monoclonal show that acceptable levels of depletion extend out to at least 24 days following a single depletion injection. Depletion of CD8 cells does not have significant effects on levels of CD4, or B-Cells (CD45R+). Acceptable levels of depletion can also be achieved with the anti-CD4 monoclonal, however, multiple depletion injections of 5-10 micrograms of antibody are required. Depletion of CD4+ cells has no obvious effects on levels of circulating CD8+ cells. At NIEHS, we have begun to examine the effects of cell depletion in specific host resistance models selected for these studies. These models include infection using bacteria (Listeria monocytogenes or Streptococcus pnuemoniae), an intracellular parasite (Plasmodium yoelii) or virus (Influenza) and a tumor challenge (PYB6 or B16F10). Three host resistance models will be evaluated for each cell subpopulation, with the selected model dependent on the specific target cell. Two PYB6 tumor challenges have been completed in mice depleted of CD8+ cells. As expected, no significant differences were observed in frequency or latency of PYB6 tumors following CD8 depletion. A PYB6 tumor study in CD4-depleted mice is in progress. The identification of chemicals that have the potential to cause injury to the immune system is of considerable public health significance, as alterations in immune function can lead to increased incidence of hypersensitivity disorders, autoimmune or infectious diseases or neoplasias. 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. In April 1998 a Workshop was held at NIEHS to establish study design to determine the sensitivity and predictability of extended histopathology as an indicator of immunotoxicity as compared with the National Toxicology Program?s functional testing battery. Standardized slide sets were generated for 10 chemicals, which had previously been evaluated for their immunotoxicity using functional tests. The histological evaluation of the slides has been completed and the data has been incorporated into a database. Dr. Russell Helms and Dr. Chris Portier, a collaborator on the original risk assessment studies, have completed the data analysis of the pathology scoring. The data indicate that for a majority of the outcomes, there was good agreement between the pathologists. However, even for variables with excellent agreement there was some evidence of inconsistency between pathologists. A direct comparison of the ratings for each pathologist indicates that, even where there was good agreement, certain individuals tended to be more conservative while others were more able to discern subtle changes than others. Additional analyses examined the consistency of a pathologist's ratings in a single tissue by investigating the correlation among all the measures in the same tissue type. When data from all pathologists were combined, measures in each of the four tissues seemed highly correlated with the other measures from that tissue. However, when correlations were examined for each pathologist independently, only the thymus evaluations maintained the same high degree of correlation. For spleen, bone marrow and lymph node measures, the outcomes for each pathologist were less well correlated, raising concern about the consistency of a pathologist's ratings across different measures in each of these tissues. A manuscript reporting these findings is in preparation.