靶向性超抗原EGF-SEA融合基因的构建及表达

目的：构建金黄色葡萄球菌肠毒素A（SEA）和表皮生长因子（EGF）表达载体。方法：利用PCR及RT-PCR技术分别克隆出SEA基因及EGF基因片断,以经过改良优化的桥式PCR将2个基因融合,再转入表达载体pET-44,经诱导剂诱导后分泌SEA-EGF融合蛋白。结果：所得SEA、EGF基因测序结果示与GENEBANK中公布的标准序列一致,且成功融合SEA-EGF基因并成功导入表达载体。结论：该研究成功构建了SEA-EGF表达载体,为进一步研究SEA-EGF融合蛋白抗头颈肿瘤靶向免疫治疗奠定了基础。     

金黄色葡萄球菌新型肠毒素基因构建与表达

目的从金黄色葡萄球菌SD和104菌株中克隆肠毒素基因,以期获得新型肠毒素。方法采用已知的金葡菌肠毒素基因保守序列设计引物,以SD和104菌株基因组为模板,PCR扩增产物插入表达载体pET28a并转化大肠埃希菌,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,镍离子金属螯合(Ni+-NTA)亲和层析纯化重组蛋白,测定重组蛋白超抗原活性及体外抑瘤效果。结果成功扩增到2个新型肠毒素P基因;带有质粒pET28a-SEPSD和pET28a-SEP104的重组大肠埃希菌,在1 mmol/L IPTG诱导下高效表达,重组蛋白纯度>95%;纯化蛋白刺激人淋巴细胞增殖率分别>30%;抑瘤率明显高于金黄色葡萄球菌肠毒素C2(SEC2),200 ng/mL时抑瘤效果最佳,分别达到82%和86%。结论成功克隆到金葡菌新型肠毒素P基因,SEPSD和SEP104重组蛋白具有与SEC2相近的活性并有较高的抑瘤效果。     

Influence of narrow-band UVB phototherapy on cutaneous microbiota of children with atopic dermatitis

OBJECTIVE: In the present work, the effect of narrow-band ultraviolet B (UVB) phototherapy on a cutaneous microbial population was evaluated in patients with atopic dermatitis (AD) and compared with control patients (vitiligo). METHODS: Count, isolation and identification of cutaneous microbiota from anticubital fossa were performed in 10 controls and 10 AD patients, both submitted to similar levels (P > 0.05) of UVB phototherapy (4.3 +/- 0.9 and 4.3 +/- 0.8 accumulated joules, respectively). Additionally, Staphylococcus aureus isolates were screened for the production of exotoxins. RESULTS: The total and staphylococcal cutaneous microbial population levels were higher (P < 0.05) in AD patients than in the controls. All these population levels decreased (P < 0.05) for both AD and control patients after UVB phototherapy, which also decreased the SCORAD for AD patients. All patients with AD and 50% of controls were carriers of S. aureus, and harboured the bacteria simultaneously on skin and anterior nares. All of the S. aureus strains recovered from AD patient skin produced toxin and the B type was the most frequently detected (70%), followed by C (20%) and A (10%) toxins. Only 40% of the S. aureus isolates from control patients produced toxin. After UVB treatment, microbial population levels of AD patients were similar (P > 0.05) to the ones found in control patients before phototherapy, and toxin production ability of S. aureus isolates decreased drastically. CONCLUSION: The results of the present study show the beneficial effect of UVB phototherapy on AD and suggest that this may be attributable not only to reduction of skin surface bacteria but also to the suppression of superantigen production from S. aureus.     

Induction of unresponsiveness to the superantigen staphylococcal enterotoxin B: cyclosporin A resistant split unresponsiveness unfolds in vivo without preceding clonal expansion

The continuous presence of antigen and powerful immune responses(exhaustive cell proliferation) of llgand reactive T cells arecurrently thought to condition clonal deletion and/or inductionof unresponsiveness to endogenous or exogenous superantigens(SAg). Here we report that in vivo induction of unresponsivenessto the SAg staphylococcal enterotoxin B (SEB) can be an immediateprocess. Within hours a large portion of ligand reactive V_ß8⁺T cells becomes clonally deleted by apoptosis. In parallel,the remaining V_ß8⁺ T cells are unresponsive to SEB,yet at the same time express functional IL-2 receptors (IL-2R)and thus are highly responsive to the growth promoting effectsof IL-2. In a subsequent step refractory IL-2R⁺V_ß8⁺T cells undergo a wave of cell proliferation for 48 h, presumablydriven by IL-2. Thereafter a large proportion of V_ß8⁺T cells succumb to apoptosis, the remaining cells display thehallmarks of split unresponsiveness, i.e.they display a selectivefallure to produce IL-2 upon SEB stimulation in vitrocombinedwith a preserved capability to express functional IL-2R. Earlydeletion and Induction of unresponsiveness to SEB are cyclosporinA (CsA) resistant, while clonal expansion with subsequent celldeletion is blocked by CsA, yet the development of split unresponsivenessis not impaired by CsA. The results suggest that IL-2 drivengrowth of refractory T cells may mimic powerful immune responsesof ligand reactive V_ß8⁺ T cells. Since unresponsivenessto SEB precedes in vivo expansion, the results as such questionthe concept of ‘exhaustive cell proliferation’ asa prerequisite for induction of unresponsiveness. In additionthey suggest that unresponsiveness (tolerance) can be inducedin the presence of CsA.     

超抗原诱导活化的细胞是Th1而不是Th2

目的：分析超抗原活化的外周血ＣＤ４＾＋Ｔ细胞Ｔｈ１／Ｔｈ２的平衡状况。方法：在体外检测了细胞因子及细胞因子受体水平。结果：ＳＥＢ能诱导外周血淋巴细胞增殖；增殖反应发生在ＳＥＢ刺激后的ｄ３；６细胞增殖达到高峰，随后下降。而ｍＩＬ－２Ｒ的表达在刺激后的ｄ３表现出明显升高（Ｐ〈０．０１），比ｓＩＬ－２Ｒ的释放提前了３ｄ。ＩＬ－２的释放在刺激后的ｄ３到ｄ４达到高峰；ＩＬ－４的水平为０。结论：早期ｍＩＬ－２     

Higher Bcl-2 levels decrease staphylococcal superantigen-induced apoptosis of CD4+ T cells in atopic dermatitis

BACKGROUND: Staphylococcal superantigens (SsAgs) contribute to the persistence of allergic skin inflammation in atopic dermatitis (AD). The aims of this study were to (1) determine whether there are differences between AD patients and healthy subjects in SsAg-induced caspase-3 activation and SsAg-induced changes of anti-apoptotic protein Bcl-2 and Bcl-2 mRNA levels of CD4+ T cells; (2) investigate the effect of interleukin (IL)-4 on SsAg-induced caspase-3 activation and SsAg-induced changes of Bcl-2 and Bcl-2 mRNA levels of CD4+ T cells. METHODS: Using immunofluorescence staining followed by flow cytometric analysis and real-time PCR, we analyzed peripheral blood mononuclear cells with or without staphylococcal enterotoxin B (SEB) stimulation in the presence or absence of recombinant IL-4 or anti-IL-4 neutralizing antibodies in 16 AD patients and 14 healthy subjects. RESULTS: SEB-reactive (TCRVbeta3+, Vbeta12+, and Vbeta17+) CD4+ T cells from AD patients were more resistant to SEB-induced caspase-3 activation and SEB-induced decrease of Bcl-2 and Bcl-2 mRNA than those from healthy subjects. Exogenously added IL-4 inhibited SEB-induced caspase-3 activation and SEB-induced decrease of Bcl-2 and Bcl-2 mRNA in SEB-reactive CD4+ T cells from healthy subjects. Inhibition of endogenous IL-4 by using anti-IL-4 neutralizing antibodies up-regulated SEB-induced caspase-3 activation and SEB-induced decrease of Bcl-2 and Bcl-2 mRNA in SEB-reactive CD4+ T cells from AD patients. CONCLUSIONS: Following SsAg stimulation, IL-4 produced by T cells in AD patients down-regulates SsAg-induced caspase-3 activation and apoptosis of CD4+ T cells through inhibiting the decrease of Bcl-2. This may impair deletion of SsAg-activated T cells and resolution of allergic skin inflammation.     

金黄色葡萄球菌超抗原与慢性鼻窦炎伴鼻息肉发病的荟萃分析

目的：慢性鼻窦炎伴鼻息肉是全球健康问题,因其易复发且症状较重,而严重影响患者生存质量。细菌超抗原学说有可能解释其发病机制。本荟萃分析旨在研究金葡菌超抗原与慢性鼻窦炎伴鼻息肉发病的关系。方法计算机检索Pubmed、MEDLINE、EMBASE、中国知网、万方数据库、CBM和VIP中关于金葡菌超抗原与慢性鼻窦炎伴鼻息肉的病例对照研究,时限为从建库到2013年1月。对纳入研究的质量进行严格评价与提取资料,对符合标准的文献进行荟萃分析。统计学分析应用RevMan 5.0软件和和GRADEprofiler 3.2.2软件。结果共纳入12篇病例对照研究。荟萃分析结果显示：病例组与对照组的鼻腔金葡菌培养阳性率[OR=5.16,95%CI（2.44,10.90）,P〈0.0001]、金葡菌超抗原阳性率[OR=11.79,95%CI（3.69,37.63）,P〈0.0001]、特异性IgE阳性率[OR=12.67,95%CI （5.08,31.61）,P〈0.00001]的差异均有统计学意义。病例组中金葡菌超抗原或抗体阳性组与阴性组嗜酸性粒细胞计数相比较,其差异没有统计学意义[WMD=36.75,95%CI（-8.09,81.60）,P=0.11]。结论金葡菌超抗原与慢性鼻窦炎伴鼻息肉具有相关性,金葡菌超抗原可能是慢性鼻窦炎伴鼻息肉发病的危险因素。