Organization of secondary lymphoid tissue and local IgE formation to Staphylococcus aureus enterotoxins in nasal polyp tissue

BACKGROUND: Bilateral nasal polyposis (NP) is characterized by high concentrations of IgE in NP tissue, which show no relation to the atopic status. We aimed to study the relationship between systemic and local IgE formation, nasal carriage of Staphylococcus aureus and nasal polyposis. METHODS: In serum and nasal tissue homogenates from 24 NP patients and 12 controls, we determined concentrations of total IgE and IgE antibodies to inhalant allergens and S. aureus enterotoxins (SAEs; A,B,C,D,E,TSST) by ImmunoCAP. Tissue cryosections were stained for CD3, CD20, CD38, CD23, FcepsilonRI, IgE and SEA/SEB. RESULTS: We demonstrated a higher incidence of S. aureus colonization (17/24) and IgE antibodies to SAEs in NP tissue (12/24) compared with controls (3/12 and 0/12, respectively). Total IgE and IgE antibodies in serum and NP tissue were dissociated because of local polyclonal IgE formation in NP tissue. Staining of NP tissue revealed follicular structures characterized by B and T cells, and lymphoid accumulations with diffuse plasma cell infiltration. CONCLUSIONS: We demonstrated the organization of secondary lymphoid tissue in polyp tissue and a polyclonal hyper-immunoglobulinemia E associated with the presence of IgE antibodies to SAEs, colonization with S. aureus, and tissue eosinophilia in a relevant subgroup of polyp patients.     

Abnormal T cell responses to bacterial superantigens in Behçet's disease (BD)

This study examines the nature of T cell hypersensitivity in BD. Highly purified T cells from 32 BD patients, from 29 rheumatoid arthritis (RA) patients and from 14 healthy individuals were cultured with various concentrations of Staphylococcal enterotoxins (SE) B and C₁ in the presence of monocytes for 5 days, after which the production of interferon-gamma (IFN-γ) was assessed. High concentrations of SE (1 ng/ml) stimulated BD T cells as well as control T cells to produce comparably high amounts of IFN-γ, whereas low concentrations of SE (1 pg/ml) stimulated BD T cells much more effectively than normal or RA T cells. The hypersensitivity of BD T cells to low concentrations of SEC₁ was restored with RA monocytes instead of BD monocytes, whereas BD monocytes could not elicit the SEC₁-induced IFN-γ production of RA T cells. Moreover, there were no significant differences between BD T cells and RA T cells in monocyte-independent IFN-γ production stimulated with low or high concentrations of immobilized anti-CD3, or in the monocyte-mediated enhancement of IFN-γ production stimulated with a low concentration of immobilized anti-CD3. These results confirm that T cell hypersensitivity is not confined to certain specific antigens in BD. More importantly, the data strongly suggest that abnormalities in signal transduction triggered by perturbation of T cell receptors, but not in that induced by cross-linking of CD3 molecules nor in that delivered through costimulation molecules, play an important role in the pathogenesis of BD.     

金黄色葡萄球菌肠毒素B基因克隆、表达及其抗血清在食物中毒快速检测上的应用

目的克隆和表达金黄色葡萄球菌肠毒素B（SEB）基因，制备金黄色葡萄球菌肠毒素B（SEB）的多克隆抗体，应用于金黄色葡萄球菌引起的食物中毒的诊断。方法用核酸扩增技术从金黄色葡萄球菌菌株中分离产生肠毒素B的标准菌株，采用高保真PCR技术从金黄色葡萄球菌野生株中扩增全长SEB，目的片段经TA克隆后DNA序列测定，重组质粒pGEM—SEB经酶切获得的SEB基因片段与真核表达载体pGEX-4T-3连接，将重组真核载体pGEX-4T-3-SEB转化人E．coli BL21（DE3）株，在0．1mmol／LIPTG诱导下，诱导SEB基因在E-coli BL21（DE3）株中表达，用谷光苷肽Sepharose4B柱分离纯化GST融合蛋白，用SDS—PAGE检测重组SEB（rSEB）的表达，重组蛋白免疫BALB／c小鼠获得抗血清，并用抗血清与天然的SEB和rSEB进行Westembolt分析。结果成功克隆了822bpSEB基因，编码272个氨基酸，在E．coli BL21（DE3）株中表达了rSEB，分离纯化的rSEB能够诱导小鼠产生IgG抗体，此抗体不仅能够与rSEB反应，而且能够与天然的SEB特异性的反应。结论重组SEB具有抗原特性，制备的特异性抗体可应用于金黄色葡萄球菌引起的食物中毒的快速诊断，将能够建立特异性强、敏感性高的金黄色葡萄球菌的诊断体系，同时也有助于对SEB在抗肿瘤机制、炎症过程中的作用等方面研究。     

低气道超敏反应中鼻源性葡萄球菌肠毒素B对Th2细胞的影响

目的探讨由葡萄球菌肠毒素B（SEB）在慢性鼻炎鼻窦炎（CRS）和哮喘之间联系的作用，并确定来源于CRS的SEB在哮喘发病机制中的作用。方法38例同时患CRS和哮喘病的病人，行鼻内镜手术外科治疗，手术后分别对血清中特异性IgE和细胞因子进行检测，并对CRS和哮喘病的临床症状进行评估，分离培养外周血单核细胞（PBMCs），存在或不存在特异性抗原和SEB的情况下，于培养的PBMCs中观察Th2反应。对照组包括：25例仅患CRS的病人，23例仅患哮喘的病人，20例健康对照。结果鼻窦手术后，同时患CRS和哮喘的病人的临床症状和肺功能有所改善，对照组为单独患CRS病人的临床症状改善。术后细胞因子、IL-4、IL-5较术前明显降低，对照组术前、术后没有明显变化。受特异性抗原刺激，培养的PBMCs产生Th2反应，而SEB在维持Th2反应所必需。对照组没有产生Th2反应。结论与鼻窦炎有关的低气道超敏反应中，细菌超抗原SEB在维持Th2反应过程中起了关键作用。     

SEC治疗胶质瘤的体外实验

目的观察SEC活化的淋巴细胞对胶质瘤的杀伤作用。方法分离并培养外周血淋巴细胞,培养液中加入不同浓度的SEC,将淋巴细胞与胶质瘤细胞(SHG-44、MGR2、MGR3、SF767、SF295、SKMG-1、T98G及UW28)混合培养,按MTT法测定淋巴细胞对瘤细胞的杀伤作用。结果经SEC活化的淋巴细胞对8株胶质瘤均产生强大的杀伤,其中以10μ/ml剂量组作用最明显。SEC作用的第一天,淋巴细胞就被激活并对胶质瘤细胞产生杀伤,第三天杀伤率达到峰值。结论SEC活化的淋巴细胞对胶质瘤细胞有明显的抗瘤活性。     

重组超抗原葡萄球菌肠毒素A基因的克隆及表达载体的构建

目的　克隆金黄色葡萄球菌肠毒素 A(SEA)全长基因并构建其表达载体。方法　以金黄色葡萄球菌的基因组 DNA为模板 ,用两条针对 SEA全长基因特异的引物 ,通过 PCR方法克隆 SEA全长基因 ,PCR产物与p GEM- T载体连接 ,经测序证实后进行亚克隆 ,构建其表达载体 p ET- 2 8b- SEA,再次测序验证。结果　克隆了 SEA全长基因 ,编码 2 5 7个氨基酸残基 ,经测序证实与 Genbank中收录的 SEA基因序列完全一致 ;并构建了 SEA的表达载体。结论　成功克隆了 SEA全长基因并构建了其表达载体 ,为进一步研究 SEA的融合蛋白的抗肿瘤活性奠定了基础     

Sensitive and specific colorimetric ELISAs for Staphylococcus aureus enterotoxins A and B in urine and buffer.

We present here simple, sensitive and accurate colorimetric capture ELISAs for staphylococcal enterotoxins A and B. Standard curves were linear over the range 0.5–1 ng/mL, and toxins could be accurately measured at 0.5 ng/mL in assay buffer or 0.1 ng/mL in human urine. Cross-reactivity between serotypes was negligible. Detection in serum was complicated by the presence of specific antibodies to SE’s in most normal sera. These assays offer a viable, cost-effective method for analysis of these ubiquitous toxins. Further, their sensitivity in undiluted urine makes them ideal candidates for evaluating human exposure.     

In vivo and in vitro adjuvant activities of the B subunit of Type IIb heat-labile enterotoxin (LT-IIb-B5) from Escherichia coli

The pentameric B subunit of the Escherichia coli LT-IIb enterotoxin (LT-IIb-B₅) activates TLR2 signaling in macrophages. Herein we demonstrate that LT-IIb-B₅, in contrast to a TLR2-nonbinding point mutant, induces functional activation of bone marrow-derived dendritic cells and stimulates CD4⁺ T cell proliferation, activities which suggested that LT-IIb-B₅ might function as an adjuvant in vivo. Indeed, in an intranasal mouse immunization model, LT-IIb-B₅ augmented specific mucosal and serum antibody responses to a co-administered immunogen, at levels which were almost comparable to those induced by intact LT-IIb holotoxin, a potent but toxic adjuvant. Therefore, LT-IIb-B₅ displays useful adjuvant properties which, combined with lack of enterotoxicity and relative stability against degradation, may find application in mucosal vaccines.