葡萄球菌肠毒素超抗原广谱抑制性多肽的设计及空间结构研究

目的:以SEs氨基酸高度保守序列作靶序列设计抑制性多肽,研究筛选出的多肽P72的空间结构。方法:用生物信息学软件"Vector NTI 10.3,Insight II 2000,Discovery Studio 1.7"等分析及预测多肽P72的空间结构。结果:P72在SEA、SEB和SEC的同源序列在空间结构具有高度的相似性,P72远离SEB的TCR和MHCⅡ结合位。结论:P72可能不是与MHCⅡ类分子及TCR结合而产生的抑制作用,其具体的抑制机制有待深入研究。     

合成多肽P72对葡萄球菌肠毒素超抗原抑制效应的研究

目的 观察合成多肽P72对葡萄球菌肠毒素超抗原(SEs)促人外周血单个核细胞(PBMC)增殖活性的抑制作用.方法 3H-TdR掺入法检测人 PBMC的增殖;ELISA检测IL-2、IFN-γ、TNF-β分泌情况.结果 P72对 SEA、SEB、SEC的促PBMC增殖及分泌IL-2、IFN-γ、TNF-β具有抑制作用.结论 细胞试验显示合成多肽P72对超抗原SEA、SEB、SEC的活性具有抑制作用.     

用间接ELISA检出葡葡球菌肠毒素B和C单克隆抗体的特异性、交叉反应性及竞争现象(文摘)

已发现七种血清学上不同的葡萄球菌肠毒素(SEA、SEB、SEC1、SEC2、SEC3SED、SEE)。它们的活性、结构和分子量均类似。在SEB和SEC间存在较多的交叉反应决定簇。该研究的目的在于建立具有仅对SEB或SEC1特异的McAbs。 采用“囊培养技术”产生毒素。SEA、SEB SEC1、SED和SEE是纯化的,SE-C2和SEC3是未经提纯的。抗血清为纯化的羊抗SEA、SED和SEE,兔抗SEB和SE-     

SED超抗原MHCⅡ类分子结合位点的研究

目的：运用定点突变技术确定SED超抗原的MHCⅡ结合位点。方法：首先检测突变体的促T淋巴细胞增殖活性；用FTTC标记SED，对增殖活性降低的突变体，进一步用竞争结合实验检测其MHCⅡ结合活性。结果：突变体SEDN23A、SEDN23A/H26R和SEDF45A的促增殖活性均显著降低，初步推测23、26和45位氨基酸可能参与了SED与MHC的相互作用。竞争结合实验证明F45位氨基酸是SED与MHCⅡ类分子结合的关键位点。结论：F45位氨基酸是SED与MHCⅡ类分子结合的关键位点；23和26位氨基酸可能参与了SED与T细胞受体(TCR)Vβ的识别，值得进一步研究。     

MHCⅡ类分子转录激活因子锤头状核酶的生物学活性

目的 探讨MHCⅡ类分子转录激活因子（CⅡTA）锤头状核酶抑制细胞表面MHCⅡ类分子的表达。方法设计并克隆针对CⅡTA第464位点的锤头状核酶（Rz464）及其相应的CⅡTA靶基因，分别插入pGEM-T载体，进行细胞外切割活性鉴定，进一步将Rz464亚克隆入真核表达载体pIRES2-EGFP（pIRES2-EGFP-Rz464，pRz464），并稳定转染Raji细胞株，流式细胞术检测经典的MHCⅡ（HLA-DR、-DP、-DQ）类抗原表达，RT-PCR分析CⅡTA mRNA水平。结果细胞外活性鉴定表明，Rz464具有明显的切割活性。细胞内切割实验显示：pRz464阳性Raji细胞表面HLA-DR、DP、DQ抗原表达分别降低了75．93％、64．14％、78．32％；同时CⅡTA的mRNA含量降低（P〈0．05）。结论抗CⅡTA锤头状核酶Rz464可有效抑制MHCⅡ类抗原的表达。     

沙眼衣原体感染对HeLa细胞MHCⅠ、Ⅱ类分子表达的影响

目的　探讨沙眼衣原体 (Chlamydiatrachomatis ,Ct)K型感染对HeLa细胞MHCⅠ、Ⅱ类分子表达的影响。方法　用免疫荧光法和流式细胞术等方法 ,对Ct感染和未感染的HeLa细胞MHCⅠ类分子表达水平和IFN γ诱导的HeLa细胞MHCⅡ类分子表达水平进行检测 ,同时对IL 10抗体的影响也作了研究。结果　Ct感染细胞MHCⅠ类分子表达水平和IFN γ诱导的感染细胞MHCⅡ类分子表达水平 ,随Ct感染剂量增加和感染时间延长而下降 ,与正常未感染细胞比较上述差异均具有统计学意义 (P <0 .0 1)。IL 10抗体能部分抑制感染细胞MHCⅠ类分子表达下调 ,但对IFN γ诱导的感染细胞MHCⅡ类分子表达下调无显著影响。结论　Ct感染可下调感染细胞MHCⅠ类分子和IFN γ诱导的细胞MHCⅡ类分子表达水平 ,这可能是造成衣原体持续感染的重要原因。感染过程中分泌的IL 10在下调感染细胞MHCⅠ类分子表达过程中起一定作用 ,而对IFN γ诱导的感染细胞MHCⅡ类分子表达水平下调无显著性影响     

MHC-I分子抗原呈递的研究进展

细胞表面的MHC-I类分子主要呈递细胞内的抗原，是细胞毒T淋巴细胞发挥免疫保护功能的重要基础。二十多年来，随着基因组学等实验技术的不断发展和新的生物信息学方法的不断提出和应用，MHC-1分子抗原呈递的研究取得了巨大进展。本文从MHC—I抗原加工呈递的分子机制、MHC—I结合多肽的来源蛋白以及MHC-I结合多肽的结构和构成特征等三个方面对MHC—I分子抗原呈递这一基本的免疫学问题进行综述。     

HLA抗原与传染性疾病免疫力

MHC 分子具有高度的多态性,不同的MHC 分子结合与呈递抗原的能力不同。由于人群中的MHC 组成千变万化,而T 细胞只能识别与Ⅰ类或Ⅱ类MHC 分子结合的抗原,因此不同的个体对同一种传染病的抵抗力强弱不等。这一假说始终未得到有力地证实。本文研究了西非人群中MHC 分子与恶性疟抵抗力之间的关系,首次报道MHC-Ⅰ类抗原(HLA-Bw53)和MHC-Ⅱ类抗原     

MHC Ⅰ类分子胞内组装、转运及其调控机制的研究进展

MHC Ⅰ类分子是启动机体免疫应答的重要分子。当细胞遭受病毒感染或本身发生癌变,MHCⅠ类分子能结合病毒或癌变细胞蛋白降解形成的多肽,向CD8+T细胞报告病毒或者肿瘤的存在,从而促使其对异常细胞的杀伤。在此过程中,MHCⅠ类分子的胞内转运途径精密调控着免疫应答的效率,但目前对MHCⅠ类分子在胞内转运途径的机制了解不多。本综述就MHCⅠ类分子在内质网的组装、外运以及内吞后降解或再循环过程中的主要机制做一阐述,并且介绍一些新发现的相关调控分子。     

HLA-G研究进展

HLA-G分子属MHC Ⅰ类非典型分子(MHC Ⅰb),组织特异性地高表达于胎母界面的滋养层细胞,MHC Ⅰ、Ⅱ类抗原是缺乏的。HLA-G分子通过NK细胞受体抑制NK细胞杀伤活性,并作为CD8~+细胞毒抑制性T细胞的识别和激活因子,抑制CTL的杀伤作用,HLA-G在胎母耐受中发挥重要作用。     

Different staphylococcal enterotoxins bind preferentially to distinct major histocompatibility complex class II isotypes

The stimulation of T cells by staphylococcal enterotoxins (SE) is strictly dependent on major histocompatibility complex (MHC) class II-bearing cells. The interaction between SE and MHC class II molecules was studied on the human B cell lymphoma Raji and its MHC class II-negative variant RJ 2.2.5. Affinity purification with SEA and SEB matrix allowed the isolation of HLA-DR-like molecules from detergent lysates of 125I surface-labeled Raji cells, but not from RJ 2.2.5 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis also revealed preferences in the binding of other SE such as SED, SEE and toxic shock syndrome toxin 1 to DR-like molecules, SEC2 to HLA-DQ-like molecules and SEC3 to DR- and DQ-like molecules. Preadsorption of the different MHC class II MHC isotypes confirmed the preferential binding of SEA to DR and of SEC2 to DQ. The implications of these findings for the understanding of SE-induced T cell activation and the potency of SE as a tool in the study of MHC class II antigens are discussed.     

Locally superantigen-activated peritoneal cytolytic T lymphocytes belong to the CD8+ CD45RC- subset and lyse MHC class II+ tumor cells.

Bacterial encoded superantigens (SA) are capable of activating and targeting cytolytic human and mouse T lymphocytes (CTL) to lyse major histocompatibility complex class II positive (MHC class II+) target cells. In this study both in vitro and in vivo activated rat CTL were directed against MHC II+ tumor targets by bacterial encoded SA. Polyclonal in vitro activation of rat peripheral blood T lymphocytes generated CTL capable of killing MHC class II+ human BSM cells coated by staphylococcal enterotoxin (SE) -A, -E, -D, and TSST-1 but not by SEB or SEC1-3. Allo selective peritoneal CTL generated by intraperitoneal stimulation with allogeneic spleen cells were directed against BSM cells by SEA, -D, and -E but not by SEB, SEC1-3 or TSST-1. Based on the above observations, and in order to locally activate CTL, SEA was chosen for in vivo priming of rats by intraperitoneal inoculation of the toxin. SEA injection generated highly cytolytic CTL, and maximum cytolytic responses were seen at 50-250 micrograms SEA per animal with a peak in response 48-72 hours after injection of the toxin. The cytolytic activity of peritoneal SEA reactive effector cells was confined to the TCR alpha beta+ CD4- CD8+ CD45RC- cell population. MHC class II- colon carcinoma cells were insensitive to lysis by SEA reactive CTL but colon carcinoma cells induced to express MHC class II by interferon-gamma (IFN-gamma) treatment were efficiently lysed in the presence of SEA. Comparison of rat and human MHC II+ colon carcinomas revealed a peak in sensitivity to lysis at 10-100 ng SEA/ml for both tumor targets. These findings suggest that superantigens can be used in local immunotherapy of peritoneal tumors such as ovarian and colorectal carcinomatosis, with inducible or constitutive expression of MHC class II.     

Induction of emetic response to staphylococcal enterotoxins in the house musk shrew (Suncus murinus)

The emetic responses induced by staphylococcal enterotoxin A (SEA), SEB, SEC2, SED, SEE, SEG, SEH, and SEI in the house musk shrew (Suncus murinus) were investigated. SEA, SEE, and SEI showed higher emetic activity in the house musk shrew than the other SEs. SEB, SEC2, SED, SEG, and SEH also induced emetic responses in this animal model but relatively high doses were required. The house musk shrew appears to be a valuable model for studying the mechanisms of emetic reactions caused by SEs.     

Involvement of HLA class II molecules in acquisition of staphylococcal enterotoxin A-binding activity and accessory cell activity in activation of human T cells by related toxins in vascular endothelial cells.

Human umbilical vascular endothelial cells (HUVEC) express HLA class II molecules upon stimulation with recombinant human interferon-gamma (IFN-gamma). Staphylococcal enterotoxin (SE) A (SEA)-binding assay using [125I]-SEA showed the presence of specific SEA binding in HUVEC stimulated with IFN-gamma but not in unstimulated HUVEC. Levels of HLA class II expression and SEA-binding increased as the IFN-gamma concentration and the period of stimulation were increased. Binding of [125I]-SEA to the IFN-gamma-stimulated HUVEC was reduced markedly by an anti-DR/DP MoAb. T cells produced IL-2 upon stimulation with a group of SEs (SEA, SEB, SEC, SED and SEE) in the presence HUVEC stimulated with IFN-gamma but not in the presence of control HUVEC. The level of accessory cell activity in the IFN-gamma-stimulated HUVEC was related to the level of HLA class II expression and SEA-binding activity. Antibodies to HLA class II molecules almost completely inhibited the response. These results indicate that HLA class II molecules are directly involved in the acquisition of these activities in HUVEC.     

Reactivity of Human γδ T Cells to Staphylococcal Enterotoxins: a Restricted Reaction Pattern mediated by Two Distinct Recognition Pathways

Staphylococcal enterotoxins (SEs) are known superantigens for T cells expressing the αβ T-cell receptor (TCR). They bind to MHC class II molecules on antigen-presenting cells and can subsequently trigger T-cell responses by binding to Vβ-gene products. The reactivity of γδ T cells with enterotoxins is less well defined although both proliferative and cytotoxic responses have been described. In the present study we have tested the cytotoxic reactivity of a panel of 41 γδ T-cell clones against target cells coated with the enterotoxins SEA, SEB, SEC1, SEC2, SEC3, SED, SEE or TSST. Three reaction patterns were observed with the γδ T-cell clones: (1) clones that specifically lysed SEA-coated target cells only; (2) clones that specifically lysed SEE-coated target cells only, and (3) clones that specifically lysed SEA-coated target cells only in the presence of certain human sera. The presence of SEA-specific antibodies in such human sera could be demonstrated. Moreover, γδ T- cell clones of this third category expressed the IgG FcRIII (CD16) which indicates that these clones are capable of mediating antibody-dependent cellular cytotoxicity towards SEA-coated target cells. Thus, the cytotoxic response of γδ T cells to SEs is mediated by two distinct pathways: an antibody-independent and an antibody-dependent pathway. The antibody-independent reactivity of γδ T cells was directed to either SEA or SEE, whereas antibody-dependent reactivity was found only towards SEA. The capacity of γδ T-cell clones to respond to stimulation with SEs, combined with their high cytolytic capacity in vitro, suggests that these cells can be involved in SE-directed immune responses and efficiently kill SE-coated target cells in vivo.     

Generation of protective immunity by inactivated recombinant staphylococcal enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity

At this time there are no vaccines or therapeutics to protect against staphylococcal enterotoxin B (SEB) exposure. Here, we report vaccine efficacy of an attenuated SEB in a nonhuman primate model following lethal aerosol challenge and identify several biomarkers of protective immunity. Initial in vitro results indicated that the mutation of key amino acid residues in the major histocompatibility complex (MHC) class II binding sites of SEB produced a nontoxic form of SEB, which had little to no detectable binding to MHC class II molecules, and lacked T-cell stimulatory activities. When examined in a mouse model, we found that the attenuated SEB retained antigenic structures and elicited protective immune responses against wild-type SEB challenge. Subsequently, a vaccine regimen against SEB in a nonhuman primate model was partially optimized, and investigations of immune biomarkers as indicators of protection were performed. SEB-naïve rhesus monkeys were vaccinated two or three times with 5 or 20 μg of the attenuated SEB and challenged by aerosol with wild-type SEB toxin. Unlike exposure to the native toxin, the vaccine did not trigger the release of inflammatory cytokines (TNFα, IL6, or IFNγ). All rhesus monkeys that developed anti-SEB serum titers ≥10⁴ and elicited high levels of neutralizing antibody survived the aerosol challenge. These findings suggest that the attenuated SEB is fully protective against aerosolized toxin when administered to unprimed subjects. Moreover, experiments presented in this study identified various biomarkers that showed substantial promise as correlates of immunity and surrogate endpoints for assessing in vivo biological responses in primates, and possibly in humans, to vaccines against SEs.     

Site of nonrestrictive binding of SEA to class II MHC antigens

We have used the synthetic peptide approach to show that the N-terminal 45-amino acids of staphylococcal enterotoxin A (SEA), SEA(1-45), constitute an important part of its binding site on class II major histocompatibility complex (MHC) molecules. SEA(1-45) and to a lesser extent SEA(1-27) were able to displace SEA from HLA-DR on Raji cells as assessed by flow cytometry and to compete with radiolabeled SEA for interaction with HLA-DR in a direct binding assay. Specific binding of SEA to Ia on murine A-20 cells could be inhibited by the same peptides [i.e. SEA(1-45) greater than SEA(1-27)] that blocked binding to HLA-DR. Therefore, different class II MHC molecules associate with the same functional site on SEA. Further, an ELISA system was used to demonstrate that SEA(1-45) is able to directly bind to a mouse synthetic I-A beta b peptide, I-A beta b (65-85), which contains a binding site of the class II MHC molecule involved in SEA presentation to T cells. Thus, we have localized a site on SEA that is involved in selective surface association with class II MHC antigens and identified the region on the class II MHC antigen to which that site binds.     

Human leukocyte antigen-DQ8 transgenic mice: a model to examine the toxicity of aerosolized staphylococcal enterotoxin B

Staphylococcal enterotoxins (SEs) belong to a large group of bacterial exotoxins that cause severe immunopathologies, especially when delivered as an aerosol. SEs elicit the release of lethal amounts of cytokines by binding to major histocompatibility complex (MHC) class II and cross-linking susceptible T-cell receptors. Efforts to develop effective therapeutic strategies to protect against SEs delivered as an aerosol have been hampered by the lack of small animal models that consistently emulate human responses to these toxins. Here, we report that human leukocyte antigen-DQ8 (HLA-DQ8) transgenic (Tg) mice, but not littermate controls, succumbed to lethal shock induced by SEB aerosols without potentiation. Substantial amounts of perivascular edema and inflammatory infiltrates were noted in the lungs of Tg mice, similar to the pathology observed in nonhuman primates exposed by aerosol to SEB. Furthermore, the observed pathologies and lethal shock correlated with an upsurge in proinflammatory cytokine mRNA gene expression in the lungs and spleens, as well as with marked increases in the levels of proinflammatory circulating cytokines in the Tg mice. Unlike the case for littermate controls, telemetric evaluation showed significant hypothermia in Tg mice exposed to lethal doses of SEB. Taken together, these results show that this murine model will allow for the examination of therapeutics and vaccines developed specifically against SEB aerosol exposure and possibly other bacterial superantigens in the context of human MHC class II receptors.     

Staphylococcal enterotoxin H contrasts closely related enterotoxins in species reactivity

Staphylococcus aureus enterotoxin H (SEH) belongs to the staphylococcal enterotoxin (SE) family of superantigens (SAgs). SEH has structural similarities to other SE; however, its biological properties are less well characterized. SEH binds with high affinity to human major histocompatibility complex (MHC) class II and exhibits strong mitogenic activity in human T cells, although it was found to be less potent than the related SEA. Surprisingly and in sharp contrast to related SEs, SEH did not possess superantigen activity in murine T cells and T cells from three investigated rat strains. However, SEH bound to a high extent to murine MHC class II expressing cells and when presented by these cells SEH stimulated human T cells to proliferate. Thus, SEH interacts with the murine MHC class II molecule in a functional manner. Notably, SEH had an inhibitory effect on murine SEA response, demonstrating that SEH interferes with the SEA interactions with murine cells. Despite this, murine T cells did not proliferate regardless of whether SEH was presented on human or murine MHC class II expressing cells. Consequently, SEH differs in species reactivity as compared to related SEs and lacks critical properties for T-cell activation in mice. We propose that unlike other SEs, SEH does not interact with murine T cells since it is not recognized by murine T-cell receptors.     

Targeting of human cytotoxic T lymphocytes to MHC class II-expressing cells by staphylococcal enterotoxins.

The staphylococcal enterotoxins (SE) comprise a family of structurally related phage-encoded bacterial proteins, which are the most potent mitogens known for murine and human T lymphocytes. In this report we describe a novel cytotoxic mechanism, where SE directs human CD3+ T lymphocytes to mediate strong cytotoxicity against target cells of irrelevant nominal specificity. The SE-dependent cellular cytotoxicity (SDCC) occurred at picomolar concentrations of SE and involved the initial binding of the SE to the target cells and subsequent triggering of the cytotoxic T cells. SDCC was induced by SEA, SEB, SEC1 and SED, which indicates that this is a common property conserved among all SE. Certain antibodies to the HLA-DR molecule efficiently blocked SDCC. Major histocompatibility complex (MHC) class II+ RAJI cells and HLA-DR-transfected murine L cells were sensitive to SDCC, whereas the MHC class II- RJ.2.2.5 RAJI cell mutant and untransfected L cells were completely resistant to SDCC. These results demonstrate that the MHC class II antigen is the target molecule in SDCC. HLA-DR molecules acted as receptors for SE and the complex was recognized by T lymphocytes in a polyclonal fashion. SDCC was mediated by allospecific cytotoxic CD8+ T cells, by cloned CD8+ T cells and by fresh human peripheral blood mononuclear cells. The SDCC phenomenon provides a rapid, potent and specific mechanism for elimination of HLA-DR+ target cells. We suggest that SDCC is an important combat strategy, employed by the bacteria to avoid specific MHC class II antigen-dependent immune recognition, by inducing T-cell dependent autologous lysis of MHC class II-expressing cells.