应用胭窝淋巴结实验评价干扰素的免疫毒性

目的 用小鼠胭窝淋巴结实验（Popliteal lymph nodeassay，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 采用小鼠胭窝淋巴结实验方法测定胭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 干扰素可引起小鼠胭窝淋巴结肿大，并造成胭窝淋巴结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，提示用胭窝淋巴结实验对干扰素（Interferon，IFN）的免疫毒性进行初步评估。     

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。     

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。     

弓形体重组质粒pcDNA_3-SAG1免疫小鼠诱导的细胞免疫应答研究

目的 :用 pc DNA3- SAG1真核表达质粒直接免疫小鼠 ,观察 DNA免疫所诱导的小鼠细胞免疫应答 ,为研制弓形虫疫苗奠定基础。方法 :大量制备重组质粒 pc DAN3- SAG1,然后将其导入 BAL B/ c小鼠体内 ,每隔 2 1d接种一次 ,一共免疫三次。用 MTT方法对脾脏的 NK细胞和其淋巴细胞的转化率进行测定 ;采用免疫荧光法对 CD4+、CD8+ 细胞进行测定。结果 :实验组 NK细胞杀伤率为 :70 .0± 3.6 4,而 pc DNA3及空白对照分别为 :48.5± 6 .0 8和 47.0± 5 .93。实验组 NK细胞活性比对照组明显增高 (P<0 .0 5 ) ,而 pc DNA3质粒及空白对照组差异无显著性 (P>0 .0 5 ) ;对 T淋巴细胞亚群 CD4+、CD8+进行动态分析 ,可见随着感染时间的延长 ,CD8+的数量逐渐上升 ,CD4+ / CD8+的比率逐渐下降 ,实验组与 pc DNA3质粒及空白对照有明显差异 (P<0 .0 5 ) ;Con A刺激小鼠淋巴细胞转化实验 ,能刺激免疫鼠及对照鼠淋巴细胞增生 ,实验组与 pc DNA3质粒及空白对照组差异无显著性 (P>0 .0 5 )。结论 :重组质粒pc DNA3- SAG1免疫 BAL B/ c小鼠可诱导一定的细胞免疫。     

艾滋病病毒外膜蛋白与干扰素融合基因表达产物的免疫学研究

为了将艾滋病病毒外膜蛋白(env)与干扰素(IFNα-2b)融合基因表达的融合蛋白作为免疫原免疫小鼠，观察小鼠的免疫功能状态和细胞免疫应答，将IFNα-2b基因片段插入到env基因的下游，经脂质体转染，筛选重组痘苗病毒，经SDS-PAGE和Western blot鉴定表达产物。用重组病毒vJ16env／IFNα-2b免疫小鼠，以生理盐水和野生型痘苗病毒作为对照，检测小鼠脾淋巴细胞对ConA、LPS及IgG的反应性；用流式细胞仪测定小鼠脾细胞CD4^+、CD8^+T细胞计数与CTL。结果表明，与对照组比较，实验组脾淋巴细胞对ConA、LPS及IgG的反应性显著增高(P&;lt;0．05)；CD4^T淋巴细胞计数和CTL活性也显著增高(P&;lt;0．05)；CD8^T淋巴细胞计数呈增高趋势，但未达到显著意义的程度。结论：重组病毒vJ16env／IFNα-2b能增强小鼠的免疫功能和诱导细胞免疫。     

银杏叶提取物对小鼠免疫功能的影响

目的:了解银杏叶提取物对小鼠免疫功能的影响。方法:采用体外抗体形成细胞检测法检测银杏叶提取物对小鼠脾细胞产生抗体的影响;采用间接免疫荧光法测定银杏叶提取物对小鼠CD4+/CD8+T淋巴细胞亚群的影响;采用3 H-TdR掺入法检测银杏叶提取物对小鼠淋巴细胞转化活性的影响。结果:银杏叶提取物有促进小鼠体液免疫功能的作用(P<0.01);能通过调整CD4+/CD8+T细胞亚群的比值来增强小鼠免疫功能(P<0.05);能增加小鼠淋巴细胞转化活性(P<0.001)。结论:银杏叶提取物能增强小鼠免疫功能。     

探讨几种中药注射剂对腘窝淋巴结免疫细胞表面分子的影响及其致敏性

目的分析中药注射剂(traditional Chinese medicineinjection,TCMI)对腘窝淋巴结免疫细胞与致敏相关的三类10余种表面分子表达的影响,探讨其用于评价TCMI致敏的可行性。方法选用♀BALB/c小鼠,选取临床上报道有过敏病例发生的痰热清等11种TCMI,后肢足趾部注射免疫动物1次,5 d后处死动物,流式细胞术检测注射侧腘窝淋巴结效应T细胞、B细胞、抗原提呈细胞及早期活化分子CD69+、MHCⅡ、协同刺激分子CD86+、CD40+等3类10余种表面分子的变化。结果痰热清、苦碟子、脉络宁注射液引起三类表面分子明显变化,具有较大的致敏潜能。柴胡、炎琥宁、刺五加等注射液所致的相应变化不明显。结论对免疫细胞三类表面分子变化的综合考察可以初步判断TCMI致敏潜能的强弱,结果与现有不良反应报道具有一致性。     

聚乙二醇化重组复合干扰素对小鼠免疫功能的影响研究

目的研究聚乙二醇化重组复合干扰素(PEG-IFNcon)对小鼠免疫功能的影响。方法以重组复合干扰素(IFNcon,商品名:干复津)为对照,分别采用鸡红细胞吞噬法和四甲基偶氮唑盐(MTT)比色法测定巨嗜细胞吞噬功能和淋巴细胞增殖作用,以3H-TdR掺入法测定刀豆蛋白A(Con A)诱导的淋巴细胞增殖活动。结果PEG-IFNcon能提高小鼠腹腔巨噬细胞吞噬功能,显著改善环磷酰胺所致的小鼠淋巴细胞功能降低作用,在50~150μg/mL剂量范围内亦能增强小鼠Con A诱导的淋巴细胞增殖反应。结论PEG-IFNcon可显著增强小鼠免疫功能。     

酵母多糖对免疫功能的影响

目的从酵母菌中提取其多糖成分,探讨酵母多糖对小鼠免疫功能的影响,为酵母多糖的临床应用提供实验依据。方法取小鼠脾脏淋巴细胞,置于96孔细胞培养板中,加入酵母多糖为酵母多糖组和细胞培养液为空白对照组,测定小鼠脾细胞的增殖活性;取细胞上清液,测定细胞因子白细胞介素-2(IL-2)、干扰素-γ(IFN-γ);用流式细胞术(FCM)检测细胞表型,观察酵母多糖对细胞免疫和体液免疫作用。结果实验组细胞增殖活性显著高于对照组(P<0.01);实验组IL-2,IFN-γ的浓度与酵母多糖在一定浓度范围内成剂量依赖关系;酵母多糖即能刺激B淋巴细胞,也能刺激T细增殖。结论酵母多糖可提高小鼠免疫功能,它不仅能提高体液免疫功能,而且能提高细胞免疫功能。     

Effects of water extraction site of Arnebia euchroma (Royal) Johnston on subgroups of T lymphocytes and haematological Indexes in mice

目的 观察新疆紫草水提部位对免疫抑制小鼠的免疫调节作用.方法 以腹腔注射环磷酰胺(CTX)制造小鼠免疫抑制模型,应用新疆紫草水提部位给小鼠连续灌胃15天,经流式细胞仪对小鼠T淋巴细胞亚群进行测定,用血细胞分析仪测定血液学指标.结果 新疆紫草水提部位能显著增加免疫低下小鼠的CD3+、CD4+T淋巴细胞的百分含量,增高CD4+/CD8+比值,降低CD8+T淋巴细胞的百分含量;同时提高血液中红细胞、白细胞、淋巴细胞计数和提高血红蛋白量、淋巴细胞百分比.结论 新疆紫草水提部位可以有效地调节机体的免疫功能.     

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.     

Positive responses to imipramine in the popliteal lymph node assay are due to primary irritation

The popliteal lymph node (PLN) assay has long been proposed as a tool to detect immunotoxicants with the potential to induce systemic autoimmunity. A major problem hampering the further validation of this assay is the need to rule out irritants that cause false-positive PLN responses. The anti-depressant, imipramine, has not been reported to induce systemic autoimmune reactions in treated patients, but has been repeatedly found positive in the PLN assay, suggesting that this is a false-positive response. To test this hypothesis, the effects of imipramine were compared to those of 50% ethanol in C57B1/6 mice. Footpad edema was evidenced in the few days after injection of both ethanol and imipramine. T-cell depletion using monoclonal antibodies against either CD4+ or CD8+ T-lymphocytes prior to the PLN assay did not influence the responses to either ethanol or imipramine. Cytokine (TNFalpha, IL-1alpha, IL-1beta, IL-2R, IL-6, IL-12 and IFN-gamma) fingerprinting of the PLNs after injection of ethanol and imipramine evidenced the same pattern of responses. These results indicate a closely similar pattern of responses following the footpad injection of either imipramine or ethanol. The conclusion can be drawn that imipramine induces positive responses in the PLN assay via primary (nonspecific) irritation.     

The IgE adjuvant effect of particles: characterisation of the primary cellular response in the draining lymph node

Diesel exhaust particles, and polystyrene particles (PSP) as a model for the insoluble particle core, have an adjuvant effect on allergen-specific IgE production in mice. We therefore examined the primary immune response in the draining popliteal lymph node (PLN) to the allergen ovalbumin (OVA) injected together with polystyrene particles into the footpad of BALB/cA mice. Similar numbers of particle-containing cells were observed in the draining lymph node on day 1 after injection of PSP alone or OVA + PSP, the numbers increasing continuously until day 21. The total lymph node cell numbers increased three to four times in the OVA + PSP group compared to both OVA and PSP groups, peaking on day 5. The increase in B cell numbers was twice the increase in T cell numbers. On day 5, OVA + PSP increased the expression of most surface markers measured (MHC class II, CD86, CD23, CD69) compared to OVA and PSP. Further, the ex vivo production of IL-4 and IL-10 by PLN cells from OVA + PSP-injected animals was increased. In conclusion, whereas PSP alone did not influence any of the immunologic markers studied, the adjuvant effect of PSP on the IgE antibody response to OVA was associated with an early increased primary cellular response in the draining lymph node.     

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.     

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.     

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。     

Immune modulation by cadmium and lead in the acute reporter antigen-popliteal lymph node assay.

Immune modulation by heavy metals may cause serious adverse health effects in humans, although the mechanisms involved are not well understood. Both cadmium and lead are important environmental and occupational toxins. Therefore, in the current study, the costimulatory/adjuvant effects and the T-cell-activating potential of these metals (i.e., CdCl2 and PbCl2), are examined. These immune-modulating properties are critical in the development of conditions such as allergy, hypersensitivity, and autoimmunity. Using the direct popliteal lymph node assay (PLNA) and reporter antigen-popliteal lymph node assay (RA-PLNA) both metals were examined individually for immunotoxicity. Mercury (i.e., HgCl2) was included for comparative purposes as its effects in the RA-PLNA are well documented. Seven days following a single footpad injection containing metal and/or RA (trinitrophenyl-ovalbumin [TNP-OVA] or TNP-Ficoll), BALB/c mice were sacrificed and the popliteal lymph nodes (PLNs) removed. PLN cellularity, TNP-specific antibody-secreting cells (ASCs), and lymphocyte subsets were assessed. All three metals strongly stimulated T- and B-cell proliferation and ASC production following coinjection with the RA TNP-OVA. In each case, ASC production was skewed towards the IgG1 isotype. In addition, all three metals induced IgG production to TNP-Ficoll (although relatively weakly in the case of Cd). These results show that each of these metals can provide adjuvant signals to promote lymphocyte proliferation and enhance adaptive immune responses to unrelated antigens. Skewing of immune responses towards T helper type 2 responses suggests that each of these metals can enhance allergic and hypersensitivity reactions to environmental antigens. Furthermore, the induction of IgG responses to TNP-Ficoll, a T-cell-independent antigen, indicates that each of these metals can activate neoantigen-specific T cells. T-cell activation by metals can lead to metal hypersensitivity and has been implicated in the development of autoimmunity. This is the first report of immune modulation by CdCl2 and PbCl2 in the RA-PLNA.     

Popliteal lymph node responses to acetone and ethanol differ from those induced by streptozotocin

The popliteal lymph node (PLN) assay was proposed to detect the potential of immunotoxicants for inducing systemic autoimmune-like reactions, but also xenobiotics that are sensitizing or exert immunostimulatory properties. Results on over 100 chemicals, mostly pharmaceuticals, are available with the PLN assay and show many correlations between rodent data and the clinical experience. A major issue is that the mechanisms involved have not been fully elucidated. In order to provide mechanistic clues to improve the predictability of the PLN assay, the effects of streptozotocin (STZ) were compared to those of ethanol and acetone in normal C57Bl/6 mice as well as mice depleted in CD4⁺ or CD8⁺ T-cells by treatment with specific monoclonal antibodies. STZ, ethanol and acetone gave similar positive responses in normal mice. Neither CD4⁺ nor CD8⁺ T-cell depletion influenced the PLN responses to ethanol or acetone, whereas CD8⁺ in contrast to CD4⁺ T-cell depletion abolished the response to STZ. There was an increase in the production of IL-6 and IFN- mRNAs measured by RT-PCR in STZ-, but not in ethanol- or acetone-treated normal mice. The production of TNF, IL-1, IL-1, IL-2R and IL-12 mRNAs was increased whatever the treatment, but increases were 2- to 3-fold greater after STZ than ethanol or acetone. These results suggest that PLN responses to primary irritants such as ethanol and acetone essentially reflect non-specific inflammation, whereas PLN responses to an autoimmunogenic compound such as STZ involve CD8⁺ T lymphocytes and the production of IFN- and IL-6. These findings may prove useful to improve the predictability of the PLN assay.