Superantigen and endotoxin synergize in the induction of lethal shock

Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD 14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro-inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100-fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin-6, and interferon-γ (IFN-γ) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D -galactosamine (D -GalN). Opposed to D -GalN-pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti-IFN-γ monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell-derived IFN-γ is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB-induced T cell deletion and anergy induction. Since Gram-positive and Gram-negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.     

T cell receptor Vβ repertoire in mice lacking endogenous mouse mammary tumor provirus

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) Vβ segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, ?7, ?8 and ?9) and 38CH (Mtv^?) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, ?7, ?8 or ?9. The TcR Vβ chain (TcR Vβ) usage in these mice was analyzed using monoclonal antibodies specific for TcR Vβ2,Vβ3, Vβ4,β5,Vβ6,Vβ7,Vβ8,Vβ11,Vβ12 and Vβ14 segments. Both Mtv-8⁺ mice and Mtv-9⁺ mice deleted TcR Vβ5⁺ and Vβ11⁺ T cells. Moreover, we also observed the deletion of TcR Vβ12⁺ cells by Mtv-8 and Mtv-9 products. Mtv-6⁺ and Mtv-7⁺ animals deleted TcR Vβ3⁺ and Vβ35⁺ cells, and TcR Vβ6⁺,Vβ7⁺ and Vβ8.1⁺ cells, respectively. Unexpectedly, TcR Vβ8.2⁺ cells were also deleted in some backcross mice expressing Mtv-7. TcR Vβ8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn → Asp) in position 19 on the TcR Vβ8.2 fragment. Reactivities of BALB.D2 TcR Vβ8.2 and 38CH TcR Vβ8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR Vβ2⁺, Vβ4⁺ and Vβ8⁺ CD4⁺ T cell subsets in Mtv-8⁺ and Mtv-9⁺ mice, and TcR Vβ4⁺ CD4⁺ T cells in Mtv-6⁺ and Mtv-7⁺ mice, when compared with the T cell repertoire of Mtv^? mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.     

Clonal anergy to staphylococcal enterotoxin B in vivo: selective effects on T cell subsets and lymphokines

Injection of bacterial superantigens such as staphylococcal enterotoxin B (SEB) in adult mice results in initial proliferation of SEB-responsive Vβ8⁺ T cells followed by induction of a state of non-responsiveness frequently referred to as clonal anergy. We show here that SEB-induced anergy involves selective changes in lymphokine production and that it affects CD4⁺ Vβ 8⁺ and CD8⁺ Vβ 8⁺ T cells in different fashions. Whereas both CD4⁺ Vβ 8⁺ and CD8⁺ Vβ 8⁺ cells from anergic mice exhibit strongly reduced proliferative capacity and interleukin(IL)-2 production upon restimulation with SEB either in vivo or in vitro the CD8⁺ subset from SEB-injected mice produces other lymphokines (such as interferon(IFN)-γ) at normal or slightly increased levels in response to SEB. Changes in the levels of production of IL-2 and IFN-γ protein correlated well with mRNA accumulation both in vivo and in vitro. Collectively these data suggest that superantigen-induced anergy involves selective changes in signal transduction and/or gene regulation in T lymphocytes.     

Ceramide-independent CD28 and TCR signaling but reduced IL-2 secretion in T cells of acid sphingomyelinase-deficient mice

Ceramide generated by lysosomal acid sphingomyelinase (aSMase) has been proposed to contribute to CD28 co-stimulatory signaling pathways. We used an aSMase-deficient mouse line ( asmase ^{− / −} ) to elucidate the role of the aSMase in splenocytes stimulated with either a combination of anti-CD3 and anti-CD28 antibodies, the lectin concanavalin A (Con A) or the superantigen staphylococcal enterotoxin B. All stimuli were shown to induce IL-2 expression, Con A additionally triggered the expression of high-affinity IL-2 receptor. However, in asmase ^{− / −} mice secretion of IL-2 was significantly reduced, whereas the intracellular IL-2 levels were elevated. Proliferation of anti-CD3/anti-CD28 or Con A-stimulated aSMase-deficient splenocytes was reduced up to 50 % after 72 h in comparison to wild-type cells. We conclude that ceramide generated by aSMase is not involved in CD28 signal transduction, but rather a perturbation of the secretory system is responsible for the impaired proliferation of aSMase-deficient splenocytes.     

葡萄球菌肠毒素B突变体基因表达和功能的初步研究

目的 应用PCR致突变基因克隆技术,获得抗原性保留,但超原毒性明显下降的葡萄球菌毒素B（SEB）突变体,用作新型淋巴细胞激活分子和超抗原疫苗的研究。方法 用PCR和PCR致变技术,从SEB标准株扩增SEB（SEB－N）和SEB突变基因（SEB－M）,分别与原核表达质粒pTrc99A重组,转化大肠杆菌JM109,经筛选获得重组质粒pTrcNb和pTrcMb,用双脱氧链终止法作序列分析。表达的SEB－N和SEB－M蛋白,经双向免疫扩散试验作抗的性鉴定后,刺激小鼠脾细胞并由ELISA法检测培养上清中IL－2的水平。结果 SEB－N150位苏氨酸（密码子ACT）非定向突变为丙氨酸（密码GCT）,SEB－M23位天冬酰胺（密码子AAT）定向突变为丝氨酸（密码子AGT）。两种突变基因表达的蛋白均与抗SEB形成明显沉淀线,与天然SEB刺激小鼠脾细胞产生IL－2水平相比,SEB-N格SEB－M突变体蛋白分别下降12．5倍和40倍。结论 获得了能表达SEB－N和SEB－M突变体蛋白的2株工程菌。表达的突变体蛋白具有良好的免疫反应性,但刺激小鼠脾细胞产生白细胞介素2的超抗原生物学活性明显下降。     

A protein A based Staphylococcus aureus vaccine with improved safety

Exposure to Staphylococcus aureus does not lead to immunity as evidenced by the persistent colonization of one third of the human population. S. aureus immune escape is mediated by factors that preempt complement activation, destroy phagocytes, and modify B and T cell responses. One such factor, Staphylococcal protein A (SpA) encompasses five Immunoglobulin binding domains (IgBDs) that associate with the Fcγ domain to block phagocytosis. IgBDs also associate with Fab encoded by V_H3 clan related genes. SpA binding to V_H3-IgM that serves as a B cell receptor results in B cell expansion and secretion of antibodies with no specificity for S. aureus. SpA crosslinking of V_H3-IgG and V_H3-IgE bound to cognate receptors of mast cells and basophils promotes histamine release and anaphylaxis. Earlier work developed a prototype variant SpA_KKAA with four amino acid substitutions in each IgBD. When tested in animal models, SpA_KKAA elicited neutralizing antibodies and protection against infection. We show here that SpA_KKAA retains crosslinking activity for V_H3-IgG and V_H3-IgE. We use a rational approach to design and test 67 new SpA variants for loss of V_H3 binding and anaphylactic activities. We identify two detoxified candidates that elicit SpA-neutralizing antibodies and protect animals from S. aureus colonization and bloodstream infection. The new detoxified SpA candidates bear three instead of four amino acid substitutions thus increasing the development of SpA-specific antibodies. We propose that detoxified SpA variants unable to crosslink V_H3-idiotypic immunoglobulin may be suitably developed as clinical-grade vaccines for safety and efficacy testing in humans.     

金黄色葡萄球菌肠毒素A抑制小鼠膀胱癌的实验研究

目的 :研究超抗原 (SAg)对膀胱肿瘤的抑癌效应。 方法 :采用已建立T739小鼠皮下可移植性膀胱肿瘤模型 60只 ,随机分为SEA组 ,BCG组和生理盐水组。观察治疗后各组的肿瘤重量和存活时间。结果 :SEA组小鼠肿瘤重量 ( 0 65± 0 .18g)明显低于生理盐水组 ( 4 2 8± 0 .86g) (P <0 0 1) ,抑瘤率为 84 7% ;小鼠存活期 ( 2 3 1± 2 .4 7d)比对照组 ( 18 6± 1.50d)延长 (P <0 0 1) ,存活延长率为 2 4 1% ;与BCG组比较 ,抑瘤率与存活延长率均无明显差异 (P >0 0 5)。结论 :超抗原做为一种新型高效的抗肿瘤生物制剂对膀胱癌有明显的抑癌效应。     

金黄色葡萄球菌超抗原在不同人群中的分子特征研究

目的了解菌血症患者血液,医护工作者、非感染性疾病入院患者以及社区健康人群前鼻腔内的金黄色葡萄球菌超抗原基因分子特征。方法用PCR和multiplex-PCR方法对277株菌的毒性休克综合征毒素-1(toxicshock syndrome toxin-1,TSST-1)和肠毒素(staphylococcal enterotoxin,SE)编码基因进行了扩增。结果 277株分离的金黄色葡萄球菌中共有142株(51%)肠毒素基因检测结果为阳性。从医院内各人群组(包括医院菌血症患者组、医护人员组和非感染性疾病入院患者组)分离菌株的阳性率在50%~59%,要比社区健康人群组阳性率(36%)高(χ2=10.86,P<0.05)。医院内各人群组分离株的SE基因主要以A型为主,而社区健康人群组的分离菌株则以D型为优势型别。另外,菌血症患者血液的金黄色葡萄球菌携带基因tsst-1的阳性率要高于其他人群组(χ2=21.91,P<0.01)。结论本研究表明不同人群分离得到的金黄色葡萄球菌超抗原分子特点具有特异性,对临床金黄色葡萄球菌的诊断治疗和流行病学研究具有一定的指导意义。     

Glomerulonephritis induced by methicillin-resistant Staphylococcus aureus infection that progressed during puerperal period

A 28-year-old Japanese woman developed fever, leg edema, purpura, and abdominal pain during the puerperal period, showing nephrotic syndrome with microscopic hematuria. At first she was thought to have Henoch-Shönlein purpura nephritis and was given steroids at another hospital. Because anasarca and massive urinary protein excretion developed, she was referred to our hospital. Renal biopsy specimens showed endocapillary proliferative glomerulonephritis with massive IgA and C3d deposition along the capillary loops and in the mesangium. A bacteriological study detected methicillin-resistant Staphylococcus aureus (MRSA) in cultures of vaginal secretions, urine, stool, and pharyngeal mucus samples. Based on the diagnosis of MRSA nephritis, anti-MRSA therapy with antibiotics was started, and MRSA became negative for each culture, and urinary protein decreased. Two months after the first renal biopsy, a second renal biopsy was performed, which revealed feeble endocapillary proliferation with mild IgA and C3d deposition in the mesangium. This case showed that the delivery procedure can cause MRSA nephritis after MRSA infection, and that steroid therapy should be avoided in the early phase of this type of nephritis.