Local Lymph Node Assay with IL-18 Endpoints

描述（由申请人提供）：目前预测接触性皮肤致敏的行业标准是小鼠局部淋巴结测定（LLNA）。LLNA的一个重要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无法正确区分强烈刺激性物质和致敏性物质。LLNA通过评估试验组与载体对照组中增殖淋巴细胞的剂量反应来评估致敏性。致敏物质诱导淋巴细胞增殖，但刺激性物质也能诱导淋巴细胞增殖，因此无法准确分类。尽管对致敏物质的过度预测听起来是可以接受的，但在公共安全监管指导方针的背景下，LLNA的这种公认的局限性导致了测试过程中的一个重大问题。目前的测试指南允许在较低浓度下进行强刺激性测试，以避免全身毒性和过度的局部皮肤刺激。限制最大测试浓度作为防止过度皮肤刺激的一种手段，允许引入假阴性结果，特别是在测试强刺激物同时也具有弱致敏性时。因此，需要一个改进的终点来实现LLNA更高的准确性。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是较老的豚鼠最大化试验（GPMT）首选的测定方法。LLNA目前被视为致敏试验的金标准。虽然无动物检测目前不被监管机构接受，但开发动物替代检测的兴趣越来越大。由于在开发新的替代测试时，LLNA被用作比较标准，因此LLNA中的任何错误预测都不可避免地会使其他测试的分析复杂化。尽管区分致敏物和刺激物的能力可以通过更广泛的分子测试来完成，但在毒理学研究中很少进行额外的测试。为此，我们将评估使用细胞因子IL-18和IL-18受体表达作为增敏剂特异性标记物来增强LLNA的预测能力。大量证据表明，IL-18是接触性皮肤致敏的重要组成部分（Antonopolous et al., 2008）。为了确定IL-18是否可以作为LLNA的潜在补充终点，我们将采用以下方法：在LLNA中测试由已知非致敏刺激物、已知假阳性刺激物和已知致敏刺激物组成的基准化学品。Aim 1将测量IL-18血清水平。目的2将确定IL-18受体是否在t淋巴细胞对增敏剂的反应中上调。目的3将确定在IL-18结合蛋白致敏过程中抑制IL-18功能的影响。如果IL-18能够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.