Local Lymph Node Assay with IL-18 Endpoints

描述（由申请方提供）：预测接触性皮肤致敏的现行行业标准是鼠局部淋巴结试验（LLNA）。LLNA的一个显著限制是假阳性的频率以及假阴性的发生。这种限制是由于LLNA无法始终正确区分强烈刺激性物质和致敏物质。LLNA通过评价试验组内增殖淋巴细胞与溶剂对照组相比的剂量反应来评估致敏性。致敏物质诱导淋巴细胞增殖，但刺激性物质也可诱导淋巴细胞增殖，从而妨碍准确分类。 尽管过度预测物质作为致敏物听起来是可以接受的，但在公共安全监管指南的背景下，LLNA的这种公认的局限性导致了测试期间的重大问题。目前的测试指南允许在较低浓度下测试强刺激物，以避免全身毒性和过度的局部皮肤刺激。限制最大试验浓度作为防止过度皮肤刺激的一种手段，会导致假阴性结果，特别是在试验强刺激性和弱致敏性时。因此，需要一种改进的端点以实现LLNA中的更高准确度。 动物试验是目前唯一公认的识别致敏物质的监管手段。10年前采用的LLNA是较旧的豚鼠最大剂量试验（GPMT）的首选试验。LLNA目前被视为致敏试验的金标准。尽管监管机构目前不接受无动物试验，但开发动物替代试验的兴趣越来越大。由于LLNA在开发新的替代检测时用作比较标准，因此LLNA中的任何错误预测将不可避免地使其他检测的分析复杂化。尽管可以通过更广泛的分子检测来区分致敏剂和刺激物，但在毒理学研究中很少进行额外的检测。为此，我们将评估使用细胞因子IL-18和IL-18受体表达作为致敏剂特异性标志物来增强LLNA的预测能力。显著的证据已经证明IL-18是接触性皮肤致敏的必要组分（Antonopolous等人，2008年）。 为了确定IL-18是否可用作LLNA中的潜在补充终点，我们将采取以下方法：将在LLNA中检测由已知非致敏刺激物、已知假阳性刺激物和已知致敏物组成的基准化学品。目的1将测量IL-18血清水平。目的2将确定T淋巴细胞上的IL-18受体是否响应于致敏剂而上调。目的3将确定在用IL-18结合蛋白致敏期间抑制IL-18功能的效果。如果IL-18可以正确区分刺激物和致敏物，这将提高测试结果的准确性，大大提高消费品的安全性。

DESCRIPTION (provided by applicant): The current industry standard for predicting contact dermal sensitization is the murine Local Lymph Node Assay (LLNA). A significant limitation of the LLNA is the frequency of false positives, as well as the occurrence of false negatives. This limitation occurs as a result of the LLNA inability to always correctly distinguish between substances that are strongly irritating and those that are sensitizing. The LLNA assesses sensitization by evaluating a dose-response of proliferating lymphocytes within a test group compared to a vehicle control group. Sensitizing substances induce lymphocyte proliferation, but irritating substances can also induce lymphocyte proliferation, thus preventing an accurate classification. Even though over-prediction of substances as sensitizers sounds acceptable, in the context of regulatory guidelines for public safety, this recognized limitation of the LLNA results in a significant issue during testing. Current testing guidelines allow for strong irritant to be tested at lower concentrations to avoid systemic toxicity and excessive local skin irritation. Limiting the maximum test concentrations as a means to prevent excessive skin irritation allows for the introduction of false negatives results, especially when testing a strong irritant that is lso weakly sensitizing. Thus there is a need for an improved endpoint to achieve greater accuracy in the LLNA. Animal testing is currently the only accepted regulatory means for identifying sensitizing substances. The LLNA, which was adopted 10 years ago, is the preferred assay over the older guinea- pig maximization test (GPMT). The LLNA is currently seen as the gold standard of sensitization testing. Although animal-free assays are currently not accepted by regulatory agencies, there is a growing interest to develop animal-alternative assays. Since the LLNA is used as a standard of comparison when new alternative tests are developed, any false predictions in the LLNA will inevitably complicate analysis of other assays. Even though the ability to distinguish sensitizers from irritants can be accomplished by more extensive molecular testing, additional testing is rarely performed in toxicological studies. To this end, we will evaluate using cytokine IL-18 and IL-18 receptor expression as a sensitizer specific-marker to enhance the prediction capability of the LLNA. Significant evidence has demonstrated that IL-18 is an essential component of contact dermal sensitization (Antonopolous et al., 2008). To determine if IL-18 can be used as a potential supplementary endpoint in the LLNA, we will take the following approaches: benchmark chemicals consisting of known non-sensitizing irritants, known false-positive irritants and known sensitizers will be tested in the LLNA. Aim 1 will measure IL-18 serum levels. Aim 2 will determine if the IL-18 receptor is upregulated on T-lymphocytes in response to sensitizers. Aim 3 will determine the effect of inhibiting IL-18 function during sensitization with the IL-18 binding protein. If IL-18 can correctly discriminate irritants from sensitizers, this will allow for improved accuracy of testing results, greatly increasing the safety of consumer products.