Fos Expression in the Rat Brain After Intraperitoneal Injection of Staphylococcus Enterotoxin B and the Effect of Vagotomy

The current study was designed to locate the neuronal activation in rat brain following intraperitoneal injection of Staphylococcus enterotoxin B (SEB) and observe the consequence of preliminary subdiaphragmatic vagotomy on SEB-induced brain Fos expression to clarify the role of the vagus nerve in sensation and transmission of abdominal SEB stimulation. The results showed that intraperitoneal SEB (1 mg/kg) induced a robust Fos expression in widespread brain areas. A significant increase of Fos immunoreactive cells were observed in the solitary tract nucleus, locus ceruleus, lateral parabrachial nucleus, ventrolateral part of central gray, medial amygdaloid nucleus, central amygdaloid nucleus, ventromedial part of thalamus, dorsomedial part of thalamus, hypothalamic paraventricular nucleus, lateral habenula, and lateral septum nucleus following SEB challenge. In hypothalamic paraventricular nucleus, in addition to the dense Fos expression in the parvocellular portion, some Fos-positive cells were also observed in the anterior magnocellular nucleus of the complex. Double immunofluorescence studies showed that these Fos-immunoreactive cells were mostly oxytocinergic. The results also showed that subdiaphragmatic vagotomy largely attenuated, but not totally abrogated, the brain Fos expression induced by abdominal administration of SEB. Our data suggest that peripheral SEB stimulation can induce activation of neurons in widespread brain areas and that the vagus plays a crucial role in transmitting the signal of abdominal immune stimulation to the brain.     

葡萄球菌肠毒素B诱导脐静脉内皮细胞凋亡机制的探讨

目的探讨金黄色葡萄球菌(金葡菌)肠毒素B诱导脐静脉内皮细胞凋亡的机制。方法将不同浓度金葡菌肠毒素B感染脐静脉内皮细胞8 h后,用流式细胞术检测细胞凋亡率,同时用比色法检测TNF-α、caspase-3及caspase-8的产生量,并检测加入TNF-α抗体、caspase-3和caspase-8抑制剂后的细胞凋亡率。结果不同浓度肠毒素B作用脐静脉内皮细胞8 h后均可诱导细胞凋亡,且TNF-α、caspase-3和caspase-8的产生量均高于对照组(P0.01);而加入TNF-α抗体、caspase-3和caspase-8抑制剂后凋亡率明显降低。结论金葡菌肠毒素B可以诱导脐静脉内皮细胞凋亡,其凋亡机制可能是通过TNF-α介导的caspase-8及caspase-3激活的外源性死亡因子受体途径。     

腹腔内注射LPS和SEB诱发Ⅰ型IL-1受体在大鼠下丘脑室旁核和视上核的表达增强

目的　为揭示脑内参与神经免疫调节过程的部位和核团。方法　大鼠腹腔内给予细菌内毒素脂多糖 (LPS)或葡萄球菌肠毒素B (SEB) ,用免疫组织化学方法观察了Ⅰ型IL 1受体在脑内表达的变化。结果　Ⅰ型IL 1受体在正常成年大鼠脑内有广泛的表达 ,隔区、视前内侧区、新皮质、海马、下丘脑室旁核、视上核、下丘脑腹内侧核、弓状核和正中隆起等部位有较多Ⅰ型IL 1受体阳性细胞。与生理盐水对照组和非免疫应激对照组 (强迫游泳 )比较 ,LPS或SEB腹腔注射后大鼠下丘脑室旁核和视上核中表达Ⅰ型IL 1受体的细胞数量显著增加 ,染色加深 (P <0 0 5 )。阳性细胞的胞浆染色面积增大 ,突起染色的长度延长。结论　下丘脑室旁核和视上核在神经免疫调节过程中可能具有重要的作用。     

从噬菌体表面展示肽库中筛选葡萄球菌B型肠毒素抑制剂

通过生物淘选,从噬菌体表面展示12肽肽库中筛选能与葡萄球菌B型肠毒素(staphylococcalenterotoxinB ,SEB)结合且能抑制其肠毒活性的特异性短肽.采用Phage ELISA和MTT鉴定所得目的肽的亲和性;根据优势噬菌体阳性克隆序列合成相应多肽.利用竞争ELISA研究合成肽与SEB单克隆抗体竞争结合SEB的情况;通过动物实验考察其抑制SEB的超抗原特性和肠毒活性情况.筛选所得短肽在一定浓度范围内可以抑制SEB对鼠脾淋巴细胞的激活;合成肽与SEB质量比为16 0∶1时,合成肽可较好地抑制SEB对乳猫的肠毒活性,并对SEB引起的小鼠致死具有明显保护作用.结果表明,初步得到了能与SEB特异结合并能抑制SEB超抗原特性和肠毒活性的短肽,为进一步研制SEB高效抑制剂奠定了基础.     

Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes

We report here the standardized conditions for stimulation of macaque whole blood samples with various protein or peptide antigens, and production of significant intracellular levels of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in CD4+ as well as CD8+ T lymphocytes. We observed significantly higher levels of TNF-alpha compared with IFN-gamma in both CD4+ and CD8+ T lymphocytes from all the macaque whole blood samples stimulated with staphylococcal enterotoxin B (SEB) as an antigen. Similarly, when whole blood samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine were stimulated with either the peptide cocktail or recombinant gp160, we observed production of significant levels of TNF-alpha by both CD4+ and CD8+ T lymphocytes. These results strongly support the utility of the whole blood cytokine flow cytometry methodology for determining antigen-specific immune responses of macaques in vaccine studies.