硫普罗宁注射液腘窝淋巴结试验研究

目的用小鼠腘窝淋巴结试验（Popliteal lymph node assay,PLNA）以及检测淋巴细胞表面分子（CD4＋/CD8＋）来评价硫普罗宁注射液的免疫毒性。方法将昆明种小鼠随机分为5组,每组12只,设阳性对照组（盐酸氯丙嗪20.83mg.kg-1BW）、阴性对照组（氯化钠注射液）和硫普罗宁注射液（3.33、13.33、53.33mg.kg-1BW）3个剂量组,采用小鼠腘窝淋巴结试验方法测定腘窝淋巴结重量及细胞指数,淋巴细胞亚群测定采用流式细胞技术法,观察不同剂量组与对照组的差别。结果小鼠分别给予不同剂量的硫普罗宁注射液后,PLN（腘窝淋巴结）重量指数、细胞指数未见统计学意义的改变,但随用药剂量的增大而表现为增加的趋势。CD4＋/CD8＋淋巴细胞的比例无明显变化。结论硫普罗宁注射液有引起小鼠淋巴结肿大、增殖细胞的趋势。     

增殖诱导配体-siRNA/类脂质体复合体的临床前安全性评价

目的考察增殖诱导配体(APRIL)-小干扰RNA(siRNA)/类脂质体复合物的毒性。方法①小鼠iv给予APRIL-siRNA/类脂质体复合物500 mg·kg-1,连续14 d称量小鼠体质量及观察死亡情况;②每只豚鼠分别于第0,7和14天左侧涂抹0.2 ml APRIL-siRNA/类脂质体复合物130 g.L-1,2,4-二硝基氯代苯10 g.L-1(阳性对照),末次接触后14 d,将各受试物0.2 ml涂于右侧,持续6 h,观察24和48 h皮肤致敏情况;③小鼠分别iv给予APRIL-siRNA/类脂质体复合物125,250和500 mg·kg-1、环磷酰胺40 mg·kg-1(阳性对照)组。间隔24 h,第2次注射后6 h处死小鼠,检测小鼠骨髓嗜多染红细胞微核率;④小鼠分别iv给予APRIL-siRNA/类脂质体复合物125,250和500 mg·kg-1、环磷酰胺(阳性对照),每天1次,连续5 d,检测小鼠精子畸形率。结果与正常对照组相比,APRIL-siRNA/类脂质体复合物500 mg·kg-1组小鼠体质量和各组织无明显变化,没有小鼠死亡,未表现急性中毒现象。APRIL-siRNA/类脂质体脂质体复合物对豚鼠皮肤无致敏性。该脂质体复合物125,250和500 mg·kg-1组小鼠骨髓嗜多染红细胞微核率分别为(1.8±1.0)‰,(1.6±0.9)‰和(2.4±1.1)‰;精子畸形率分别为(1.98±0.27)%,(1.96±0.15)%和(2.04±0.16)%,与对照组微核率(2.2±1.3)‰和精子畸形率(1.96±0.29)%无明显差异。结论在本实验剂量范围内,APRIL-siRNA/类脂质体复合物对小鼠无明显毒性。     

幼年注射氟西汀诱导制备成年ICR小鼠抑郁模型

目的通过幼年注射氟西汀(Flu)制备成年小鼠抑郁模型,探讨其表观有效性和预测有效性。方法 幼年ICR小鼠出生后第4到第21天连续ip给予Flu 10 mg.kg-1(幼年诱导模型组),正常饲养至9周龄后,分为模型组、ig给予Flu 10 mg.kg-1和氟哌啶醇0.1 mg.kg-1组,连续3周后开始检测实验,测试期间继续给药,测试完成后小鼠为12～13周龄。开场实验检测跨格数,悬尾实验检测不动时间,明暗穿箱实验检测明暗穿箱次数。结果 与正常对照组相比,幼年诱导模型组小鼠成年后,自主活动显著减少(跨格数:83±30 vs 58±19;站立数:32±10 vs 20±8),悬尾不动时间显著延长〔83±46 vs(128±56)s〕,明暗穿箱次数显著减少(18±5 vs 10±4)。小鼠连续ig给予Flu 10 mg.kg-1后,自主活动显著增加(跨格数:58±19 vs 85±41;站立数:20±8 vs 30±12),悬尾不动时间显著缩短〔128±56 vs(71±40)s〕,明暗穿箱次数显著增加(10±4 vs 17±7),各指标均恢复至正常对照组水平;而给予氟哌啶醇0.1 mg.kg-1组小鼠,各指标数据无明显改变,同时表现出镇静作用。结论 幼年小鼠注射Flu成年后的表现符合抑郁模型表观有效性和预测有效性特征,有望成为制备更合理的抑郁症动物模型方法。     

宾赛克嗪拮抗M和N胆碱受体的特征及其抗胆碱酯酶抑制剂的毒性效应

目的评价宾赛克嗪(benthiactzine)对胆碱受体功能拮抗作用及其对有机磷农药中毒的抗毒效应,并与阿托品比较。方法①以烟碱诱发小鼠惊厥,离体豚鼠回肠收缩为评价中枢和外周神经元N受体功能的指标;以槟榔碱诱发小鼠震颤为评价中枢M受体功能的指标;以槟榔碱和毛果芸香碱诱发小鼠流涎,乙酰胆碱和氧化震颤素诱发回肠收缩为评价外周M受体功能的指标;并在烟碱和毛果芸香碱联用诱发混合性震颤模型上,研究药物的药理学特点。②在DDVP染毒小鼠模型上研究药物的抗毒效应(PR值)。结果①宾赛克嗪抗槟榔碱致小鼠中枢性震颤的ED50±L95为(0.31±0.06)mg.kg-1,中枢抗M作用比阿托品(1.33±0.17)mg.kg-1强3.3倍。②宾赛克嗪抗槟榔碱和毛果芸香碱诱发小鼠流涎的ED50±L95分别为(0.38±0.09)mg.kg-1和(5.57±1.30)mg.kg-1,而阿托品分别为(0.17±0.03)mg.kg-1和(0.46±0.18)mg.kg-1,宾赛克嗪的外周抗M作用弱于阿托品。③宾赛克嗪抗烟碱诱发惊厥的ED50±L95为(2.87±0.76)mg.kg-1,具有较强的中枢抗N作用。④宾赛克嗪抗烟碱诱发回肠收缩的IC50±L95为(1.1±0.18)μmol.L-1,比阿托品(13.84±1.68)μmol.L-1强约13倍,说明宾赛克嗪具有外周神经元N受体拮抗作用。⑤宾赛克嗪抗毛果芸香碱和烟碱联用诱发小鼠震颤的ED50±L95为(2.64±0.35)mg.kg-1,比阿托品(4.95±0.65)mg.kg-1强约1倍。⑥宾赛克嗪和阿托品抗DDVP致死作用的ED50±L95分别为(2.87±0.40)mg.kg-1和(6.78±3.92)mg.kg-1;宾赛克嗪10 mg.kg-1可使小鼠抗DDVP中毒的PR值提高16.84倍,而阿托品仅能提高0.81倍。结论宾赛克嗪中枢抗M作用强于阿托品,外周抗M作用弱于阿托品,并具有中枢和外周神经元N受体拮抗作用;宾赛克嗪对敌敌畏中毒小鼠的抗毒效应明显强于阿托品。     

原花青素对醋酸所致小鼠疼痛模型的影响

目的 观察原花青素对醋酸所致小鼠疼痛模型的影响。方法 取小鼠72只,随机分为6组各12只,即原花青素高、中、低剂量组（分别灌胃给予原花青素120.0,24.0,4.8 mg·kg-1）和0.9%氯化钠溶液对照组（灌胃给予等量0.9%氯化钠溶液）、吗啡对照组（吗啡5 mg·kg-1灌胃）和阿司匹林对照组（阿司匹林500 mg·kg-1灌胃）,均给药5 d。第5天所有小鼠腹腔注射0.6%醋酸溶液致痛,记录小鼠15 min内扭体次数,比较各组扭体次数。结果原花青素高、中剂量组小鼠平均扭体次数分别为（4.33±2.43）和（6.50±2.91）次,均显著少于0.9%氯化钠溶液组（均P＜0.05）;原花青素低剂量组平均扭体次数（12.83±3.74）与0.9%氯化钠溶液组差异无显著性。原花青素各剂量组与阿司匹林组［（1.83±0.72）次］和吗啡组［（0.75±0.45）次］比较,扭体次数均显著较高（均P＜0.05）。阿司匹林组和吗啡组平均扭体次数均较0.9%氯化钠溶液组少（均P＜0.05）。原花青素高、中、低剂量组扭体反应的抑制率分别为74.53%,61.76%和26.46%。阿司匹林组和吗啡组扭体反应的抑制率分别为92.16%和95.59%。结论原花青素对醋酸所致小鼠疼痛模型有明显的镇痛作用,且镇痛作用与剂量呈量效关系。     

百合地黄汤加甘麦大枣汤抗抑郁作用实验研究

目的 研究百合地黄汤加甘麦大枣汤抗抑郁作用.方法 昆明小鼠40只, 随机分为模型组、高剂量药物组、低剂量药物组及阳性对照组, 每组10只.阳性对照组灌胃给予盐酸氯米帕明溶液 0.04 g.kg^-1,高、低剂量药物组分别给予百合地黄汤加甘麦大枣汤(BG) 30, 15 g.kg^-1, 均按20 mL.kg-1体质量灌胃给予相应药物, 模型组给予同容量0.9%氯化钠溶液, 连续灌胃给药7 d.采用小鼠悬尾、强迫游泳实验及利血平拮抗实验,观察百合地黄汤加甘麦大枣汤对小鼠抑郁模型的影响.结果 百合地黄汤加甘麦大枣汤高剂量组、低剂量组、阳性对照组、模型组悬尾不动时间分别为(49.80±39.52), (49.20±33.10), (24.90±28.71), (79.40±25.17)s;游泳不动时间分别为(78.00±37.92), (81.70±34.59), (32.6±36.82), (113.9±19.81) s; 眼睑开始下垂时间分别为(18.00±6.25), (10.30±2.87), (12.40±4.67),( 6.20±3.00) s; 眼睑下垂评分(2.30±1.34),( 2.50±1.08),( 2.50±1.65),( 4.00±0.00)分.百合地黄汤加甘麦大枣汤能显著缩短小鼠悬尾和游泳不动时间,可对抗利血平所致小鼠眼睑下垂.结论 百合地黄汤加甘麦大枣汤有较好的抗抑郁作用.     

LW-AFC对环磷酰胺处理小鼠免疫功能的影响

目的探讨LW-AFC对免疫功能低下小鼠是否有改善作用。方法昆明小鼠ip给予环磷酰胺(CTX)0.02g·kg-1,连续10d,制备免疫功能低下小鼠模型。六味地黄(LW)浓缩丸(1.4g·kg-1)和LW-AFC(0.2,0.4和0.8g·kg-1)组小鼠在每次给予CTX后ig给予相应药物,连续10d。监测小鼠体重,以及胸腺和脾脏重量变化,并计算胸腺和脾脏系数;[3H]TdR掺入法检测脾淋巴细胞增殖反应,MTT法检测脾脏自然杀伤(NK)细胞杀伤活性,流式细胞术检测脾淋巴细胞中CD4+CD8-和CD4-CD8+T细胞亚群百分率,并计算CD4+/CD8+T细胞亚群比值。结果与正常对照组比较,模型组小鼠胸腺和脾重量及其系数明显降低,体重增长显著缓慢;LW和LW-AFC对小鼠体重增长缓慢、胸腺重量及其系数降低无明显改善作用;LW-AFC0.2g·kg-1对CTX导致的脾重及系数降低有明显改善(P<0.05)。模型组小鼠脾淋巴细胞自发增殖反应、刀豆蛋白A(ConA)和脂多糖(LPS)诱导的脾细胞增殖反应与正常对照组比较均明显降低,[3H]TdR掺入值由正常对照组的6115±441,19432±1778和(23345±7296)cpm分别降低为2741±340,9210±1387和(3983±263)cpm;与模型组比较,LW-AFC0.8g·kg-1对脾淋巴细胞自发增殖反应有明显的改善作用;LW浓缩丸和LW-AFC对ConA诱导的脾细胞增殖反应均具有明显的促进作用,[3H]TdR掺入值分别为13996±5161,27550±2356,15427±1444和(27333±1701)cpm;对LPS诱导的脾细胞增殖降低无改善作用,甚至低于CTX模型组(P<0.01)。模型组小鼠NK细胞杀伤活性由正常对照组的(39.5±0.5)%降低为(37.0±1.0)%,LW和LW-AFC明显促进小鼠NK细胞杀伤活性,杀伤率分别为(40.9±0.6)%,(39.7±0.8)%,(42.4±0.5)%和(39.8±0.9)%。与正常对照组比较,模型组小鼠脾脏CD3+,CD4+CD8-和CD4-CD8+T细胞百分率明显增加,CD4+/CD8+比值无明显变化;LW浓缩丸和LW-AFC可使CTX导致的升高的CD3+,CD4+CD8-和CD4-CD8+T细胞百分率明显降低,并可明显降低CD4+/CD8+比值。结论 LW-AFC对CTX所致小鼠免疫功能低下具有一定的改善作用。     

胍基琥珀酸促进小鼠学习和记忆的作用(英文)

采用水迷宫实验和一次性被动回避实验 (跳台法和避暗法 ) ,研究了胍基琥珀酸 ( 14,2 8和 56mg·kg- 1,ip)对小鼠学习和记忆的影响 .在水迷宫实验中 ,给小鼠ip胍基琥珀酸 56mg·kg- 1,每日 1次 ,连给 4d ,明显改善小鼠的记忆功能 .无论 1次或连续 4dip上述 3个剂量的胍基琥珀酸对正常小鼠一次性被动回避实验均无明显影响 ,但能明显对抗樟柳碱 ( 5mg·kg- 1,ip) ,亚硝酸钠 ( 12 5mg·kg- 1,sc)和 10 %乙醇 ( 10mL·kg- 1,po)所引起的记忆障碍 .胍基琥珀酸的上述作用近似谷氨酸 ( 30 0mg·kg- 1,ip) .结果表明 ,胍基琥珀酸类似谷氨酸和其他兴奋性氨基酸 ,在调节学习和记忆过程中可能起一定的作用     

复方壳聚糖对消化功能的影响

目的 观察复方壳聚糖给小鼠不同剂量对消化功能的影响。方法 取体重18～22gICR小鼠50只,雌雄各半,随机分为5组,分别灌胃给予蒸馏水,复方壳聚糖100、200、400mg·kg-1,云芝多糖240mg·kg-1,实验前分别灌胃炭未阿拉伯胶混悬液01mg·10g-1,15min后将动物处死测定炭末推进百分率。结果 蒸馏水组为66.49±7.22;复方壳聚糖组分别为92.73±7.08、95.32±5.72、99.35±1.72(P均 ＜0.01),云芝多糖组为79.75±10.98。结论 复方壳聚糖可显著提高小鼠胃肠的蠕动能力。     

青藤碱对吗啡依赖小鼠位置偏爱效应的影响

目的 :利用条件性位置偏爱实验研究中药成分青藤碱对小鼠吗啡奖赏效应的影响。方法 :实验采用有倾向性程序。吗啡 (9mg· kg-1,sc,每日 1次 ,5 d)引起小鼠显著的位置偏爱效应。在训练阶段每天 sc吗啡前3 0 min预先给予青藤碱 (1 0 ,3 0和 60 mg· kg-1,ip)可剂量依赖性地抑制吗啡引起的小鼠位置偏爱效应。结果 :提示预先给予青藤碱能消除吗啡产生的条件性位置偏爱。     

腘窝淋巴结免疫细胞多种表面分子在PLNA评价致敏性中的综合分析

目的利用腘窝淋巴结实验(popliteal lymph node as-say,PLNA)分析腘窝淋巴结免疫细胞多个表面分子在PLNA评价致敏性中的综合变化,旨在提高PLNA的可靠性和灵敏性。方法选用♀BALB/c小鼠,选取具有致敏潜能物质氯化汞(HgCl2)、链脲佐菌素(STZ)、2,4,6-三硝基苯磺酸(TN-BS)和D-青霉胺(D-pen)为供试品,后肢足趾部注射免疫动物1次,5 d后处死动物,取注射侧腘窝淋巴结制备细胞悬液,流式细胞术检测腘窝淋巴结淋巴细胞、效应T细胞、抗原提呈细胞及其MHCⅡ、协同刺激和早期活化等十余种表面分子的变化,探讨这些表面分子在供试品致敏后的综合改变。结果 HgCl2和TNBS引起腘窝免疫细胞多种表面分子明显变化,D-pen和STZ所致的相应变化不明显。结论腘窝淋巴结免疫细胞表面分子变化的综合分析可以判断供试品的致敏潜能;不同供试品引起表面分子变化的种类和强弱有所不同,在致敏性评价中应综合考察各细胞表面分子的变化。     

Evaluation of the use of reporter antigens in an auricular lymph node assay to assess the immunosensitizing potential of drugs.

Immune-mediated idiosyncratic drug reactions are a major problem for susceptible patients, physicians, and the pharmaceutical industry. Validated screening tools to assess the immunosensitizing capacity of orally or intravenously administered pharmaceuticals are currently not available. To date, the popliteal lymph node assay (PLNA) seems the most promising tool for this purpose. The PLNA has recently been extended with the use of reporter antigens (RA) that are coinjected together with the drug of interest. The measurement of isotypes of RA-specific antibody-secreting cells (ASC) enables the distinction of sensitizing chemicals and (nonsensitizing) irritants without radio-isotopic end points. However, the use of footpad injections raises ethical concerns. Therefore, we examined the use of RA after intradermal injection into the ear of BALB/c mice and measured RA-specific ASC in the draining auricular lymph node (ALN). We show that RA-specific IgG isotype ASC numbers are very useful and sensitive parameters to identify drug-induced hypersensitivity in both PLN and ALN. However, the type 1-associated parameters (CD8(+) cells, macrophages, IFN-gamma, TNF-alpha, and IL-1 beta) that are induced in the PLN by streptozotocin were less pronounced in the ALN. Thus, the PLNA may provide more immunologically relevant information on the mechanisms of certain chemical-induced hypersensitivity reactions. The RA-ALN assay may provide an alternative for the RA-PLNA; both assays can be used to distinguish sensitizing compounds from nonsensitizing ones.     

Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice

It has yet to be established whether chlorogenic acid (CGA), a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs). The primary limitation in evaluating this potential relationship is the lack of an effective animal model for use in predicting the immunosensitizing potential of low molecular weight compounds (LMWCs). Currently, the popliteal lymph node assay (PLNA) is considered a very promising tool for assessing immunosensitizing potential of LMWCs. To determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both a direct PLNA (d-PLNA) and a reporter antigen PLNA (RA–PLNA) method. Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP–OVA or TNP–Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.     

Increased production of interferon-gamma, but not IL-4 mRNA, by streptozotocin in the popliteal lymph node assay

The popliteal lymph node (PLN) assay has been proposed as a tool to predict systemic autoimmune reactions induced by medicinal products and chemicals, the mechanisms of which are poorly understood. To determine whether PLN responses involved Th1 or Th2 cell control, or both, the effects of streptozotocin (STZ), a prototypic immunotoxic compound, were analysed on the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) mRNA by lymph node cells after injection into the hind footpad of C57 BL/6 mice. Streptozotocin induced a dramatic increase in IFN-gamma mRNA production, which correlated with PLN responses as evidenced by augmented weight and cellularity indices. No effect on IL-4 mRNA synthesis was noted. These results suggest that a Th1 response is involved in the PLN response to STZ.     

应用腘窝淋巴结实验评价干扰素的免疫毒性

目的用小鼠腘窝淋巴结实验(Popliteallymphnodassay,PLNA)以及检测淋巴细胞表面分子来预测干扰素的免疫毒性,探讨该方法的预测价值。方法采用小鼠腘窝淋巴结实验方法测定腘窝淋巴结重量及细胞指数,淋巴细胞亚群测定采用流式细胞技术法。结果干扰素可引起小鼠腘窝淋巴结肿大,并造成腘窝淋巴结CD4+/CD8+T细胞比例改变。结论本实验中干扰素的测试结果呈阳性,提示用腘窝淋巴结实验对干扰素(Interferon,IFN)的免疫毒性进行初步评估。     

Differentiation of responses to allergenic and irritant compounds in mouse popliteal lymph node assay

The mouse popliteal lymph node assay (PLNA) has been proposed as an immunotoxicological test to predict allergenic chemicals. However, PLN response is also observed in association with non-specific activation induced by some irritants. We, therefore, examined the kinetics of the PLN cellularity indices of primary and secondary responses. Flow cytometric analysis was used to measure the proportions of T and B cells in PLN. Male ICR mice were subcutaneously injected with TNBS (an allergenic compound) or SDS (an irritant compound) in the left hind footpad, and with vehicle in the right one. On day 28 after first injection, mice were injected with 1/10 or 1/100 dose of the initial injection. On day 7 after first injection of TNBS, primary response reached maximal PLN cellularity index (16.5). On day 2 after second injection, secondary response reached maximal PLN cellularity index (13.1). A marked increase in proportion of B cells was observed in the PLN. On the other hand, after first injection of SDS, primary response reached maximal PLN cellularity index (2.8) on day 10, but neither secondary response nor increase in the B cell proportion were observed. These results suggest that the detection of secondary response and the flow cytometric analysis are useful in differentiating responses to allergenic and irritant compounds in the PLNA.     

Oxazolone and diclofenac-induced popliteal lymph node assay reactions are attenuated in mice orally pretreated with the respective compound: potential role for the induction of regulatory mechanisms following enteric administration

The murine popliteal lymph node assay (PLNA) was examined as a preclinical assay with the potential to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with immune-mediated drug hypersensitivity reactions (IDHRs) in humans. We hypothesized that the contact sensitizer oxazolone (OX) would cause a strong PLN reaction in naive mice and that the PLN reaction would be attenuated in mice orally pretreated with OX due to the induction of oral tolerance. In naive mice, OX induced a strong PLN reaction and caused dose-dependent increases in PLN size, weight, cellularity, percentage of CD4(+) PLN T cells, and percentage of PLN B cells, with a concomitant decrease in the percentage of CD8(+) PLN T cells. Next, the PLNA was conducted in mice gavaged three times with either OX or vehicle alone (olive oil). Mice pretreated with OX had suppressed PLN reactions following the footpad injection of OX (decrease in PLN size, weight, and cellularity), which was associated with an increase in the percentage of PLN CD8(+)T cells. In contrast, oral pretreatment with OX had no observable effect on the PLN reaction induced following footpad injection of the irrelevant hapten dinitrochlorobenzene (DNCB). Adoptive transfer studies were conducted to examine the mechanism of PLN hyporesponsiveness. It was found that either (1) unfractionated splenocytes or (2) purified CD8(+) splenocytes, but not (3) purified CD4(+) splenocytes isolated from mice gavaged with OX adoptively transferred PLN suppression to naive BALB/c mice. Because OX is not a pharmaceutical, we also examined the NSAID diclofenac (DF) (Voltaren). Like OX, DF caused dose-dependent increases in PLN size, weight, and cellularity in naive mice. Furthermore, like OX, the diclofenac-induced PLN reaction was attenuated in mice that had been orally pretreated three times with DF. However, splenocytes from mice orally treated with DF were not able to adoptively transfer PLN hyporesponsiveness. Collectively, these observations demonstrate that both OX and DF are potent immunostimulators in the PLNA. As importantly, these results demonstrate that the immunostimulating potential of OX and DF in the PLNA is significantly decreased in mice orally exposed to the respective drug, possibly due to the presence of a cellular mechanism of oral tolerance. For OX, the mechanism appears to involve, in part, CD8(+) T cells, whereas the mechanism(s) associated with PLN hyporesponsiveness using DF remain to be defined.     

肌肉免疫DC-EBV-LMP2诱导免疫应答的研究

目的探讨肌肉免疫DC-EBV-LMP2诱导的免疫效果。方法从BALB/C小鼠分离骨髓细胞,诱导培养获得小鼠DC,以100 MOI的rAd-LMP2感染获得DC-EBV-LMP2,继续培养48h,诱导DC-EBV-LMP2成熟。用免疫酶法确定LMP2在DC中表达。0,2,4周,以2×105个DC-EBV-LMP2/只免疫BALB/C小鼠,于第5,8周分别LMP2特异性水平和T细胞亚群的变化。结果结果显示,肌肉免疫DC-EBV-LMP2可诱导BALB/C鼠LMP2特异性免疫应答,且第5周强于第8周。CD8+T/CD3+T细胞亚群的比值在第5周高于对照组,而第8周低于对照组。结论 DC-EBV-LMP2肌肉免疫BALB/C鼠可诱导LMP2特异性CTL。     

Development of an oral exposure mouse model to predict drug-induced hypersensitivity reactions by using reporter antigens.

The capability of certain drugs to cause immune-mediated drug hypersensitivity reactions in susceptible individuals has initiated a search for pre-clinical screening tools to identify immunosensitizing drugs. Since most drugs are taken orally, hazard assessment of their immunosensitizing potential should include oral exposure models. In this study, the predictive value of the reporter antigen (RA) approach was investigated in combination with oral or intraperitoneal (ip) exposure to a selection of allergenic drugs, i.e., D-penicillamine (D-Pen), Diclofenac (DF), or Nevirapine (Nevi). The RA trinitrophenyl-Ovalbumin (TNP-OVA) was used to assess the capacity of the drugs to stimulate systemic immune responses to a bystander antigen, whereas the RA TNP-Ficoll was used to indicate whether the drugs were able to induce specific anamnestic T-cell responses. TNP-OVA was injected (ip) in C3H/HeOuJ mice that were subsequently exposed (orally or ip) to one of the drugs via different exposure protocols. All three model drugs used resulted in delayed type hypersensitivity reactions to TNP-OVA after ip and oral exposure. In addition, TNP-specific serum antibody levels were increased after ip exposure to Nevi, and after both oral and ip exposure to D-Pen and DF. These data indicate that the present drugs are able to stimulate immune responses to bystander antigens. Responses to TNP-Ficoll were measured in the popliteal lymph node of BALB/c mice three weeks after they received a single oral dose of D-Pen or DF. Results of this approach show that orally pre-treated mice responded with enhanced responses (TNP-specific IgG1 and IFN-gamma production) to sub-optimal doses of D-Pen or DF in a drug-specific manner. Data with TNP-Ficoll indicate that these drugs stimulate systemic formation of specific T cells. Together, the RA-approach allows assessment of systemic sensitization upon oral and/or ip exposure to the selected drugs. To further evaluate the utility of these models, more drugs, including non-allergenic drugs and those that require metabolic conversion to become allergenic need to be studied in the present models.     

Diclofenac activates T cells in the direct popliteal lymph node assay and selectively induces IgG(1) and IgE against co-injected TNP-OVA

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently associated with immune-mediated hypersensitivity reactions. The NSAID diclofenac is associated with several distinct allergic and autoimmune-like reactions including anaphylaxis, idiosyncratic hepatotoxicity and autoimmune hemolytic anemia. The aim of this study was to examine the immunostimulating potential of diclofenac in the direct popliteal lymph node assay (PLNA) and reporter antigen PLNA. In BALB/c mice, diclofenac caused dose-dependent increases in PLN weight and PLN cellularity in the direct PLNA; 0.25 mg was non-immunostimulating whereas 0.50-1.00 mg caused a significant PLN reaction. In the direct PLNA, diclofenac also increased the percent of T cells in the PLN with activated phenotypes (CD44(high)CD62L(low) and CD44(high)CD62L(high)). Finally, the magnitude of the diclofenac-induced direct PLN reaction was significantly reduced when the assay was conducted in T-cell-deficient mice. When co-injected with the reporter antigen TNP-Ficoll (trinitrophenyl Ficoll), 0.50 mg diclofenac caused significant increases in PLN weight, PLN cellularity, and induced IgM and IgG(1) anti-TNP antibody forming cells (AFCs) in the PLN. In a final set of studies, a TNP-OVA PLNA was conducted using diclofenac, phenobarbital (negative control) and streptozotocin (positive control). As expected, phenobarbital (1.00 mg) failed to cause an increase in PLN cellularity or induce AFCs in the PLN. Streptozotocin (1.00 mg) caused significant increases in PLN cellularity, IgM AFCs, and selectively induced IgG(2a) and IgG(2b) AFCs against TNP-OVA. Likewise, diclofenac caused dose-dependent increases (0.25-1.00 mg) in PLN cellularity and IgM AFCs. However, in contrast to streptozotocin, diclofenac caused a selective dose-dependent increase in both IgG(1) and IgE AFCs. Finally, an increase in the intracellular level of IL-4, but not INFgamma, was detected in CD4(+) PLN cells following the injection of diclofenac mixed with TNP-OVA. Collectively, these data suggest that diclofenac: (i) induces a T-cell-dependent direct PLN reaction that; (ii) provides non-cognate help for IgG AFC production when co-injected with TNP-Ficoll, possibly through the formation of neo-antigens; and (iii) possesses intrinsic adjuvant activity that selectively induces IL-4 mediated production of IgG(1) and IgE against co-injected TNP-OVA.