Potential Immunoinflammatory Role of Staphylococcal Enterotoxin A in Atopic Dermatitis: Immunohistopathological Analysis and in vitro Assay

BACKGROUND: The underlying mechanism of atopic dermatitis (AD) exacerbated by Staphylococcus aureus has not been established. However, we demonstrated recently that the majority of S. aureus strains colonized in the skin of Korean AD patients carried genes encoding staphylococcal enterotoxin A (SEA) and/or toxic shock syndrome toxin-1 (TSST-1). OBJECTIVE: To clarify the role of staphylococcal superantigen, SEA in AD. METHODS: With the lesional skin of 9 AD patients and normal looking skin of one healthy adult, we examined first the expression of SEA, staphylococcal enterotoxin B (SEB), and TSST-1 using immunohistochemical analysis. In addition, we investigated the effects of SEA on the expression of inflammation-related adhesion molecules and cytokines in human HaCaT keratinocytes and Human Umbilical Vein Endothelial Cells (HUVECs) by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and enzyme-linked immunosorbent assay. RESULTS: Staphylococcal protein A (SPA) and SEA were detected with increased immunoreactivity in AD patients. However, TSST-1 showed mild-to-moderate immunoreactivity in AD patients, whereas SEB was minimally detected. In the double immunofluorescence investigation, SEA and SPA were well co-localized. SEA induced upregulation of adhesion molecules and elicited inflammatory responses in HaCaT keratinocytes and HUVECs. CONCLUSION: This study demonstrates the importance of SEA as an immunoinflammatory triggering factor of AD in Koreans.

Preliminary studies of the toxcicity of staphylococcal enterotoxin A liposomes / 医学研究生学报

Objective: The bacterial superantigen staphylococcal enterotoxin A (SEA) has the potent ability of inducing T cell activation as well as directing activated T cells to kill tumour cells. However, its application in the tumour treatment is limited due to its systemic immune activation.In order to minimize its side effects SEA liposomes is prepared and their toxcicity in vivo is investigated . Methods: SEA liposomes were prepared by the method of reverse-phase evaporation . SEA liposomes were administered intravenously . In vivo the toxcicity of SEA liposomes was investigated by the measurement of blood pressure, colonic temperature and breath rate of New Zealand rabbits. Mice plasma TNF-? and IFN-? level were determined by ELISA . Results:After iv administration of SEA liposomes, significant reduction of mice plasma TNF-? and IFN-? level was observerd. As compared to free SEA, liposomal SEA had less effect on blood pressure, colonic temperature and breath rate of the rabbits. Conclusion:SEA Liposomes had relatively low toxicity. These advantage was probably due to its lower systemic immune activation effect and inducing consequent lower systemic TNF-? and IFN-? level.

CD28 and CTLA-4 expressions in the process of mice' T-cell inefficiency induced by staphylococcal enterotoxin A / 解放军医学杂志

Objective To explore the characteristics and regulations of CD28 and CTLA-4 expressions in the process of mice' T-cell inefficiency induced by staphylococcal enterotoxin A(SEA).Methods Twelve mice were averagely divided into three groups(4 each).The mice in group 1 received single injection of SEA,and the mice in group 2 and 3 received SEA injection twice and three times,respectively,with a 3 days interval.Mice were sacrificed at day 1,3,7 and 14 after the last injection,and then the splenic lymphocytes were isolated.The expressions of CD28 and CTLA-4 in cellular membrane and the intracellular expressions of CTLA-4 and IL-2 were detected with flow cytometry.Results In group 1,the CD28 expression in cellular membrane and the intracellular expression of IL-2 were significantly up-regulated,while the CTLA-4 expressions both in cellular membrane and intracellular expression were lower with no obvious changes.In group 2,the expressions of both CD28 and IL-2 were up-regulated slightly,the expressions of CTLA-4 increased significantly both in cellular membrane and intracellular expression,even more increase was the intracellular CTLA-4 expression.In group 3,the expressions of CD28 and CTAL-4 in cellular membrane and the intracellular CTLA-4 expression declined,and at day 7 the intracellular IL-2 expression was undetectable.Conclusions SEA may obviously promote the activation of naive T-cells when initially used to induce the splenic lymphocytes,while multiple stimulations(e.g.3 times) of SEA may lead T-cells to anergy.On the process of inefficiency,the declined IL-2 production may be closely related to the down-regulation of CD28 expression;the up-regulation of CTLA-4 expression may profit inducing inefficiency,but is not on the maintenance of inefficiency.

Growth Inhibition of Tumor by Recombinant SEA / 中国肿瘤生物治疗杂志

Objective:To evaluate the antitumor activity of recombinant SEA for therapy of B16 melanoma established in C57BL/6 mice. Methods:C57BL/6 mice with melanoma were treated with the purified rSEA. The tumors were isolated and weighted. Results:Tumor growth was apparently inhibited by rSEA at high, middle, and low doses intraperitone-ally, whose inhibition ratio were 79.3% , 75.6 % and 73. 8% respectively. rSEA treatment in situ could inhibit tumor growth more effectively(90.6% ). Further study showed that numerous CD8+ and CD4+ T cell were infiltrated in tumor tissues, which were consistent with tumor growth inhibition induced by rSEA. Conclusions: rSEA could inhibit tumor growth effectively, especially the treatment in situ. This study paves the way for tumor immunotherapy with targeted SEA.

Construction and identification of prokaryotic expression system of staphylococcal enterotoxin A gene and expressed product / 中国病理生理杂志

AIM:To construct a prokaryotic expression system of staphylococcal enterotoxin A(SEA)gene and determine the effects of the recombinant expression product rSEA in promoting lymphocyte proliferation and inhibiting tumor cell growth.METHODS:PCR was used to amplify entire SEA gene of S.aureus strain ATCC13565.The cloned SEA gene was sequenced after T-A cloning.SDS-PAGE was applied to measure the output of rSEA expressed by pET32a-SEA-E.coli BL21DE3.Ni-NTA affinity chromatography was performed to extract rSEA.Cytotoxicity of rSEA to Vero cells was detected using TCID_ 50 titration method and then the value of TCIC_ 50 was determined.MTT colorimetry was established to examine the effects of rSEA at different dosages on proliferation of mouse splenocytes and human peripheral blood mononuclear cells(PBMC)as well as on growth of HepG2 cells and HeLa cells in vitro.RESULTS:In comparison with the published corresponding sequences,similarities of the nucleotide and putative amino acid sequences of the cloned SEA gene were 100%.The output of rSEA was approximate 25% of the total bacterial proteins.rSEA had a cytotoxicity with TCIC_ 50 of 3.14 ?g to Vero cells.1.0-20.0 mg/L rSEA showed the significant effects of promoting proliferation of mouse splenocytes and human PBMC(P

The design and prediction of biology characteristics of recombinant toxin CD80-Linker-SEA / 中国免疫学杂志

Objective:To construct the eukaryotic expressed vector which express recombinant toxin CD80 Linker SEA and predict the rationality and feasibility of the linker Methods:Utilize the sequence analysis software to analyze the flexibility、antigenicity、Hoop&Woods hydrophilicity and episode of recombinant toxin CD80 Linker SEA Results:Through the analysis of the software,it could be found that the recombinant toxin has correct domains of CD80 and SEA The linker has low episode、low antigenicity and high flexibility Conclusion:The results of computer analysis could help us to rationally design the recombinant toxin CD80 Linker SEA and keep it's maximum biological activity