A murine local lymph node assay for the identification of contact allergens

The development of an alternative predictive test for the identification of contact sensitizing chemicals is described. The method is based upon the fact that, following epicutaneous application, sensitizing chemicals initiate a primary immunological response in the draining lymph node(s) which is characterized by lymphocyte proliferation. Experimental conditions for the measurement in vitro of the induced lymph node cell proliferative response have been optimized. On the basis of the data presented a local lymph node assay was developed in which CBA/Ca strain mice were exposed daily, for 3 consecutive days, to various concentrations of the test chemical, or to vehicle alone, on the dorsum of the ear. Lymph node activation was measured subsequently as a function of increased node weight, the frequency of large pyroninophilic cells and lymphocyte proliferation in the presence or absence of an exogenous source of interleukin 2 (IL-2). The results of a validation study are reported in which 22 well-characterized sensitizing chemicals of varying potency were examined. With the exception of three chemicals where water was used as the application vehicle, positive responses, defined as a substantial increase in lymphocyte proliferative activity, were recorded with all these test materials. Under the conditions employed non-sensitizing chemicals, including non-sensitizing irritant chemicals, failed to influence the immunological status of the draining lymph node. Taken together, the data suggest that the local lymph node assay provides the basis for a rapid and cost-effective alternative to the currently available guinea pig predictive test methods. The local lymph node assay may be of particular value for the evaluation of coloured or irritant chemicals.     

Arsenic-induced alterations in the contact hypersensitivity response in Balb/c mice

Previous studies in our laboratory indicate that arsenic alters secretion of growth promoting and inflammatory cytokines in the skin that can regulate the migration and maturation of Langerhans cells (LC) during allergic contact dermatitis. Therefore, we hypothesized that arsenic may modulate hypersensitivity responses to cutaneous sensitizing agents by altering cytokine production, LC migration, and T-cell proliferation. To investigate this hypothesis, we examined the induction and elicitation phases of dermal sensitization. Mice exposed to 50 mg/l arsenic in the drinking water for 4 weeks demonstrated a reduction in lymph node cell (LNC) proliferation and ear swelling following sensitization with 2,4-dinitrofluorobenzene (DNFB), compared to control mice. LC and T-cell populations in the draining lymph nodes of DNFB-sensitized mice were evaluated by fluorescence-activated cell sorting; activated LC were reduced in cervical lymph nodes, suggesting that LC migration may be altered following arsenic exposure. Lymphocytes from arsenic-treated animals sensitized with fluorescein isothiocyanate (FITC) exhibited reduced proliferative responses following T-cell mitogen stimulation in vitro; however, lymphocyte proliferation from nonsensitized, arsenic-treated mice was comparable to controls. Arsenic exposure also reduced the number of thioglycollate-induced peritoneal macrophages and circulating neutrophils. These studies demonstrate that repeated, prolonged exposure to nontoxic concentrations of sodium arsenite alters immune cell populations and results in functional changes in immune responses, specifically attenuation of contact hypersensitivity.     

进展期中上部胃癌脾门淋巴结转移与微转移的分析

目的 分析进展期中上部胃癌脾门淋巴结转移与微转移情况。方法 回顾性分析2011年8月至2014年8月82例接受全胃切除D2根治术的进展期中上部胃癌患者临床病理资料,运用免疫组化检测淋巴结微转移,分析脾门淋巴结转移和微转移的临床病理高危因素。结果 82例患者共检及150枚脾门淋巴结,其中18例发生转移(21.9%),常规病理学检测阴性的64例患者中有21例出现微转移(32.8%)。单因素及多因素分析均显示TNM分期、Borrmann分型、肿瘤横向部位是脾门淋巴结转移的高危因素,而T分期、肿瘤横向部位是微转移的独立危险因素。结论 中上部进展期胃癌脾门淋巴结转移及微转移发生率较高,Borrmann分型、TNM分期、肿瘤横向部位、T分期是脾门淋巴结总体转移的高危因素,含有以上临床病理特征者建议常规行脾门淋巴结清扫。     

Assessment of cumulative allergen-activated lymph node cell proliferation using flow cytometry.

The murine local lymph node assay (LLNA) is a method for the prospective identification of chemical contact allergens. The current validated protocol assesses lymphocyte proliferation induced in the draining lymph node as a function of in situ incorporation of radiolabeled thymidine. We have explored the potential utility of an alternative nonradioisotopic marker of cell division, the cytoplasmic dye carboxyfluoresein succinimidyl ester (CFSE). Using this method, the cell phenotype and the number of divisions each cell has undergone can be tracked using flow cytometry. BALB/c strain mice were exposed topically to various concentrations of the contact allergens 2,4-dinitrochlorobenzene (DNCB), oxazolone (ox) or hexyl cinnamic aldehyde (HCA), or to the nonsensitizing skin irritant methyl salicylate (MS). Five days later, lymph node cells (LNC) were labeled with CFSE, cultured for 96 h, then incubated with fluorescent labeled anti-CD4 (T helper) and -CD8 (T cytotoxic) cell antibodies, and proliferating CD4+ and CD8+ cells analyzed by flow cytometry. In LNC populations derived from vehicle-treated animals, less than 1% of either cell population had undergone one cell division or more. Topical exposure to MS (2.5 to 20%) did not increase the frequencies of proliferating cells. Exposure to all three allergens, however, resulted in a marked increase in the percentages of both CD4+ and CD8+ cells undergoing division, with up to 5% and 3% of these cells, respectively, proliferating in response to DNCB and oxazolone, and with lower levels of proliferation stimulated by HCA. These preliminary data suggest that this method may be applied to provide the basis of a nonradioisotopic end point for the LLNA, particularly for the identification of potent contact allergens.     

腘窝淋巴结试验模型的构建及在清开灵注射液致敏性研究中的应用

目的构建直接和间接腘窝淋巴结试验(d-PLNA和s-PLNA)模型,并用其检测清开灵注射液(QKLI)的致敏性。方法雌性BALB/c小鼠右侧后肢足趾一次性分别sc给予50μl盐酸D-青霉胺(D-Pen)12.5,25.0,37.5,50.0和62.5 mg.kg-1,分别在给药后的第5,7和9天处死小鼠,摘取两侧腘窝淋巴结(PLN),计算PLN质量指数(MI)和细胞指数(CI),确定D-Pen的最低有效剂量和最佳解剖时间。小鼠右侧后肢足趾一次性sc给予D-Pen 37.5 mg.kg-1、QKLI原液、2倍和4倍QKLI原液,7 d后活杀,进行d-PLNA实验。小鼠右侧后肢足趾一次性sc给予D-Pen和QKLI,2个月后,给予亚剂量激发,7 d后活杀进行s-PLNA实验,检测MI和CI。结果根据MI≥2和CI≥5阳性标准判定D-Pen最低有效剂量为37.5 mg.kg-1,最佳解剖时间为给药后第7天。d-PLNA实验结果显示,QKLI原液组处理侧PLN的质量和细胞计数较未处理侧有轻度的升高,但未达到阳性反应判定标准;2倍原液浓度组MI为2.0±1.0,CI为5.4±0.9;4倍原液浓度组MI为3.4±0.4,CI为5.4±0.9,均达到阳性反应判定标准。s-PLNA实验结果显示,2倍原液浓度组MI为2.4±0.6,CI为6.2±0.8;4倍原液浓度组MI为3.2±0.9,CI为8.4±1.8均达到阳性反应判定标准。结论制备了能够用于致敏实验的腘窝淋巴结模型,利用此模型发现QKLI具有诱发过敏反应的可能。     

宝石能谱CT成像在鉴别不同病理类型肿瘤及其转移淋巴结中的作用

目的探讨宝石能谱CT成像在不同组织来源、病理类型肿瘤及其转移淋巴结中的鉴别诊断价值。方法回顾性分析肺癌、胃癌和食管癌患者30例,其中包括转移淋巴结41枚,行能谱cT扫描并检测肿瘤原发病灶及转移淋巴结的cT值、碘（水）基含量、有效原子序数、能谱衰减曲线及其斜率等参数。采用独立样本t检验对各组间数据进行统计学分析比较。结果除肺鳞癌转移淋巴结外,不同病理类型肺癌原发灶之间、原发灶与转移淋巴结之间以及转移淋巴结之间的主要能谱特征性参数均未见统计学差异;但它们与胃癌和食管癌转移淋巴结在中低能量区CT值、碘（水）基含量和曲线斜率皆有显著统计学差异。肺鳞癌转移淋巴结与其它肿瘤原发灶及转移淋巴结之间在中低能量区下的CT值和碘（水）基含量的差异比较皆具显著性统计学意义。结论比对分析肿瘤原发病灶及其转移淋巴结能谱CT特征参数,对不同组织来源、病理类型肿瘤及其转移淋巴结的鉴别诊断有着一定的临床指导意义。     

肺癌患者肺门纵隔淋巴结的合理清扫方式与范围探讨

目的：探讨肺癌患者肺门纵隔淋巴结的合理清扫方式与范围。方法：选择2006年1月－2008年1月于本院手术治疗的肺癌患者45例,回顾性分析了其病例资料,分析术前胸部CT和MRI对肺门纵隔淋巴结转移的判断与术后病理结果的一致性．同时比较系统采样病例胸内各区淋巴结的转移频度,以及肺门纵隔淋巴结系统采样与单纯采样术的阳性发现率。结果：患者术前CT诊断和术后病理对肺门纵隔淋巴结转移的结果的一致性进行检验,κ为0．351;MRI诊断一致性检验．κ为0．449,优于CT。距离肺门和肺根部最近的11、10、7、5、4区淋巴结的转移频度最高,距离肺根部较远的9、6、3、2、1区淋巴结的转移频率较低。肺门纵隔淋巴结系统采样40例,阳性发现率为85．0％;单纯采样10例,阳性发现率为60．0％,两组间比较差异有统计学意义（χ^2=13．28,P〈0．05）。结论：胸部CT和MRI不应作为术前肺癌N分期的唯一检查,应引入进一步的其他诊断技术,以提高对肺门纵隔淋巴结分期的准确性。在肺癌术中应主动清除肺门和纵隔各区淋巴结,特别是围绕肺门或肺根部周围的淋巴结,不能忽略淋巴结转移的现象。系统淋巴结采样术较适合肺癌的根治性手术治疗。     

I期非小细胞肺癌病灶中nm23的表达与淋巴结微转移的关系

目的探讨I期非小细胞肺癌原发癌灶nm23基因表达与淋巴结微转移之间的关系。方法对40例根治性切除的Ⅰ期非小细胞肺癌患者的原发癌灶石蜡标本采用免疫组化方法进行nm23基因表达蛋白检测,对常规病理检查阴性的淋巴结应用免疫组化方法以Ck为标志物进行微转移检测,所得数据进行统计学分析。结果(1)本组40例非小细胞肺癌中nm23蛋白阳性表达57.5%(23/40),阴性表达42.5%(17/40)。(2)常规病理检查阴性的淋巴结192枚,有11例患者中的28枚淋巴结检出微转移灶,淋巴结微转移阳性检出率14.6%(28/192),有淋巴结微转移患者检出率27.5%(11/40)。(3)nm23蛋白阳性表达组淋巴结微转移患者发生率13.04%,nm23蛋白阴性表达组淋巴结微转移患者发生率47.06%,两组阳性率的比较有统计学意义(P<0.05)。结论Ⅰ期非小细胞肺癌患者病灶中nm23的表达和淋巴结微转移关系密切,联合检测原发癌灶中nm23的表达和淋巴结微转移灶不仅能够评估肺癌侵袭转移的程度,而且对正确评估肺癌的病理分期、预后及制定合理的治疗方案有重要意义。     

Further experience with the local lymph node assay using standard radioactive and nonradioactive cell count measurements

In a previous study, the predictive capacity of a modified local lymph node assay (LLNA) based on cell counts, the LNCC, was demonstrated to be closely similar to that of the original assay. In addition, a range of substances, including some technical/commercial materials and a range of agrochemical formulations (n = 180) have also been assessed in both methods in parallel. The results in the LNCC and LLNA were generally consistent, with 86% yielding an identical classification outcome. Discordant results were associated with borderline data and were evenly distributed between the two methods. Potency information derived from each method also demonstrated good consistency (n = 101), with 93% of predictions being close. Skin irritation was observed only infrequently and was most commonly associated with positive results; it was not associated with the discordant results. Where different vehicles were used with the same test material, the effect on sensitizing activity was modest, consistent with historical data. Analysis of positive control data indicated that the LNCC and LLNA displayed similar levels of biological variation. When taken in combination with the previously published results on LLNA Performance Standard chemicals, it is concluded that the LNCC provides a viable non‐radioactive alternative to the LLNA for the assessment of substances, including potency predictions, as well as for the evaluation of preparations. Copyright © 2012 John Wiley & Sons, Ltd.     

淋巴结病理学检查在药物安全性评价中的特点和意义

淋巴结(LN)是重要的外周免疫器官,免疫应答发生的场所。LN形态结构的改变可敏感提示药物免疫毒性的有无,表明LN毒性病理学评价是免疫毒性筛选的有力手段。本文综述了LN复杂的生理结构和毒性病理学变化特点,并阐述了LN病理检查在药物安全性评价中的作用和意义。     

Experience with local lymph node assay performance standards using standard radioactivity and nonradioactive cell count measurements

The local lymph node assay (LLNA) is the preferred test for identification of skin-sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. To facilitate acceptance of nonradioactive variants, validation authorities have published harmonized minimum performance standards (PS) that the alternative endpoint assay must meet. In the present work, these standards were applied to a variant of the LLNA based on lymph node cell counts (LNCC) run in parallel as a control with the standard LLNA with radioactivity measurements, with threshold concentrations (EC3) being determined for the sensitizers. Of the 22 PS chemicals tested in this study, 21 yielded the same results from standard radioactivity and cell count measurements; only 2-mercaptobenzothiazole was positive by LLNA but negative by LNCC. Of the 16 PS positives, 15 were positive by LLNA and 14 by LNCC; methylmethacrylate was not identified as sensitizer by either of the measurements. Two of the six PS negatives tested negative in our study by both LLNA and LNCC. Of the four PS negatives which were positive in our study, chlorobenzene and methyl salicylate were tested at higher concentrations than the published PS, whereas the corresponding concentrations resulted in consistent negative results. Methylmethacrylate and nickel chloride tested positive within the concentration range used for the published PS. The results indicate cell counts and radioactive measurements are in good accordance within the same LLNA using the 22 PS test substances. Comparisons with the published PS results may, however, require balanced analysis rather than a simple checklist approach.     

Potency values from the local lymph node assay: Application to classification,labelling and risk assessment

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: ‘extreme’, ‘strong’, ‘moderate’ and ‘weak’. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment.     

超声对甲状腺癌颈部淋巴结转移的诊断价值

目的探讨彩色多谱勒超声在甲状腺癌颈部淋巴结转移中的诊断价值。方法回顾性分析72例经手术病理证实为甲状腺癌患者的颈部淋巴结术前超声资料,记录肿大淋巴结分区、大小、二维声像图特征及内部血流状态等并与术后病理结果进行对照研究。结果转移性淋巴结超声检查与病理结果对照符合率为77.9%。淋巴结的长径/短径比值（L/S）〈2、高回声淋巴门结构消失、内部见微钙化、囊性变及丰富血流预示淋巴结转移。转移性淋巴结组与良性增生性淋巴结组上述各指标比较,差异有统计学意义（P〈0.05）。转移性淋巴结组收缩期峰值流速（PSV）和阻力指数（RI）均明显高于良性增生性淋巴结组（P〈0.05）。结论高频彩色多普勒超声是诊断甲状腺癌颈部淋巴结转移的简便、有效的方法,诊断准确性较高,对临床有重要的指导意义。     

Safety assessment of allergic contact dermatitis hazards: an analysis supporting reduced animal use for the murine local lymph node assay

The original Organisation for Economic Co-operation and Development Test Guideline 429 (OECD TG 429) for the murine local lymph node assay (LLNA) required five mice/group if mice were processed individually. We used data from 83 LLNA tests (275 treated groups) to determine the impact on the LLNA outcome of reducing the group size from five to four. From DPM measurements, we formed all possible four- and five-mice combinations for the treated and control groups. Stimulation index (SI) values from each four-mice combination were compared with those from five-mice combinations, and agreement (both SI<3 or both SI ≥ 3) determined. Average agreement between group sizes was 97.5% for the 275 treated groups. Compared test-by-test, 90% (75/83) of the tests had 100% agreement; agreement was 83% for the remaining eight tests. Disagreement was due primarily to variability in animal responses and closeness of the SI to three (positive response threshold) rather than to group size reduction. We conclude that using four rather than five mice per group would reduce animal use by 20% without adversely impacting LLNA performance. This analysis supported the recent update to OECD TG 429 allowing a minimum of four mice/group when each mouse is processed individually.     

Intracellular expression of cytokines and granzyme B in auricular lymph nodes draining skin exposed to irritants and sensitizers

The murine local lymph node assay (LLNA) has been extensively utilized to evaluate sensitizing chemicals. However, there have been some concerns that its use to discriminate between classes of chemicals is minimal. It is thus desirable to identify better or alternative immune endpoints with in LLNA itself. Here, we evaluated the protein and/or mRNA levels of cytokines and granzyme B (GzmB), a cytotoxic lymphocyte product, to discriminate between sensitizers and irritants and to characterize the chemical sensitizers when used as supplemental indicators in LLNA endpoints. For this, CBA/N mice were topically treated daily with a well-known chemical sensitizer such as a strong contact sensitizer 1-chloro-2,4-dinitrobenzene (DNCB), a skin contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (OXA), and a skin or respiratory sensitizer toluene 2,4-diisocyanate (TDI), and the non-sensitizing irritants, croton oil (CRO) and nonanoic acid (NA), for 3 consecutive days. The protein and/or mRNA levels in auricular lymph nodes draining the ear skin were then analyzed by real-time RT-PCR and immunoassay. The sensitizers, but not the irritants, evoked pronounced interleukin (IL)-2, IL-3 and IL-4 or interferon (IFN)-gamma. Significantly, different sensitizers evoked different cytokine patterns of IL-4 and IFN-gamma, as DNCB strongly up-regulated both IFN-gamma and IL-4, OXA up-regulated IFN-gamma strongly but IL-4 weakly, and TDI up-regulated IL-4 strongly but IFN-gamma weakly. The sensitizers also strongly up-regulated GzmB mRNA, while the irritants had a much weaker effect. Thus, these cytokines and GzmB mRNA may be useful as additional endpoints for discriminating between irritants and sensitizers or contact and respiratory sensitizers in the LLNA.     

Evaluation of CD86 expression and MHC class II molecule internalization in THP-1 human monocyte cells as predictive endpoints for contact sensitizers

The aim of this study was to explore the usefulness of a human monocyte cell line in the development of in vitro models for predictive testing of contact sensitizers. Several studies have shown that contact sensitizers induce CD86 expression and enhanced internalization of MHC class II molecules in dendritic cells (DCs). We used THP-1, a human monocyte cell line, as a replacement for DCs for evaluation of these phenotypical alterations as predictive endpoints for contact sensitizers. Known sensitizers and irritants were evaluated. After 24-h exposure to samples, the expression of CD86 on THP-1 cells was measured by flow cytometry. Sensitizers such as dinitrochlorobenzene (DNCB), 2-mercaptobenzothiazole (MBT), eugenol, p-phenylenediamine (PPDA) and ammonium tetrachloroplatinate (Pt) enhanced CD86 expression on THP-1 cells, while nickel sulfate, cobalt sulfate and irritants such as methylsalicylate (MS), sodium dodecyl sulfate (SDS) and dimethyl sulfoxide (DMSO) did not augment CD86 expression. A synergistic effect was observed when DNCB and IFN- were added simultaneously to a culture of THP-1 cells. Furthermore, internalization of MHC class II molecules was observed when the cells were treated with some of sensitizers for 2 h. The inducing effects of chemicals on the two phenotypical alterations were the same. These results suggest that these test systems can be used to predict contact-sensitizing ability of chemicals as an in vitro sensitization assay.     

Classification of contact allergens according to potency: proposals

It is clear that contact allergens vary substantially with regard to the relative potency with which they are able to induce skin sensitisation. Considerations of potency will in the future become a significant factor in the classification of skin sensitising chemicals. It is therefore appropriate to establish what is known of potency and thresholds in the induction of skin sensitisation and the elicitation of allergic contact dermatitis, and to identify approaches that might be available for assessment of relative potency for the purposes of categorising chemical allergens. This paper was prepared by an ECETOC (European Centre for Ecotoxicology and Toxicology) Task Force that had the objective of recommending approaches for the measurement of potency and definition of thresholds for both the induction and elicitation of contact sensitisation. The deliberations recorded here build upon recommendations made previously by an ECETOC Task Force that considered the conduct of standard skin sensitisation test methods for the purposes of hazard identification and risk assessment (ECETOC, Monograph No. 29, Brussels, 2000). The emphasis in this present paper is also on standard and accepted methods for the assessment of skin sensitisation, and for which OECD guidelines are available: the local lymph node assay (LLNA), the guinea pig maximisation test and the occluded patch test of Buehler. For various reasons, discussed in detail herein, attention focused primarily upon consideration of categorisation of chemical allergens and the identification of thresholds with respect to the induction of skin sensitisation, rather than the elicitation of allergic contact dermatitis. It is concluded that although the LLNA is the method of choice for the determination of skin sensitisation potency for the purposes of categorisation, if data are already available from appropriate guinea pig tests then their judicious interpretation may provide information of value in determinations of potency and categorisation. Included here are detailed and specific recommendations for how best the results of the three test methods considered can be used for the categorisation of chemical allergens as a function of skin sensitisation potency.     

Targeted delivery of docetaxel to the metastatic lymph nodes: A comparison study between nanoliposomes and activated carbon nanoparticles

The objective of this study is to compare the targeting ability of activated carbon nanoparticles and nanoliposomes, which are used as carriers for delivering docetaxel (DTX) to the metastatic lymph nodes. In this study, we first prepared the DTX-loaded activated carbon nanoparticles (DTX-AC-NPs) by modifying the activated carbon with nitric acid oxidation and absorbing DTX in the concentrated nitro-oxide nanocarbon. We then prepared DTX-loaded nanoliposomes (DTX-LPs) by the proliposome method. The physiochemical properties of DTX-AC-NPs and DTX-LPs were carefully evaluated in vitro. The metastatic lymph node uptake and the injection site retention were investigated by analyzing the DTX concentration in metastatic lymph nodes and injection sites. The result showed that DTX-AC-NPs and DTX-LPs with suitable and stable physicochemical properties could be used for in vivo lymph node targeting studies. DTX-AC-NPs significantly increased DTX-AUC_(0–24) and prolonged DTX-retention in metastatic lymph nodes compared to DTX-LPs and non-modified activate carbon in vivo. This study demonstrated activated carbon nanoparticles may be potential intralymphatic drug delivery system to preferentially target regional metastatic lymph nodes.     

B cell increases and ex vivo IL-2 production as secondary endpoints for the detection of sensitizers in non-radioisotopic local lymph node assay using flow cytometry

Non-radioisotopic local lymph node assay (LLNA) using 5-bromo-2′-deoxyuridine (BrdU) with flow cytometry (FCM) is gaining attention since it is free from the regulatory issues in traditional LLNA (tLLNA) accompanying in vivo uses of radioisotope, ³H-thymidine. However, there is also concern over compromised performance of non-radioisotopic LLNA, raising needs for additional endpoints to improve the accuracy. With the full 22 reference substances enlisted in OECD Test Guideline No. 429, we evaluated the performance of LLNA:BrdU-FCM along with the concomitant measurements of B/T cell ratio and ex vivo cytokine production from isolated lymph node cells (LNCs) to examine the utility of these markers as secondary endpoints. Mice (Balb/c, female) were topically treated with substances on both ears for 3 days and then, BrdU was intraperitoneally injected on day 5. After a day, lymph nodes were isolated and undergone FCM to determine BrdU incorporation and B/T cell sub-typing with B220⁺ and CD3e⁺. Ex vivo cytokine production by LNCs was measured such as IL-2, IL-4, IL-6, IL-12, IFN-γ, MCP-1, GM-CSF and TNFα. Mice treated with sensitizers showed preferential increases in B cell population and the selective production of IL-2, which matched well with the increases in BrdU incorporation. When compared with guinea pig or human data, BrdU incorporation, B cell increase and IL-2 production ex vivo could successfully identify sensitizers with the accuracy comparable to tLLNA, suggesting that these markers may be useful for improving the accuracy of LLNA:BrdU-FCM or as stand-alone non-radioisotopic endpoints.