新型截短肠毒素C2突变株抑制肿瘤细胞生长

在恶性肿瘤的治疗中,肠毒素C2 (SEC2) 的临床应用由于其副作用而被严重限制。利用SEC2基因截短技术,获得保留T细胞刺激活力又不引发催吐效应的SEC2突变株,可有效解决这个问题。根据噻唑蓝比色法 (MTT) 分析结果,新型截短肠毒素C2突变株 (NSM) 可显著刺激T细胞增殖,并且可显著抑制人大肠癌细胞 (CX-1) 和人乳腺癌细胞 (MCF-7) 生长。NSM的T细胞刺激能力和抑瘤效果与SEC2相似。动物研究结果证明NSM不再引发呕吐效应,且可显著抑制荷瘤小鼠的肿瘤生长。因此,这种可抑制肿瘤细胞生     

Superantigen-induced apoptotic death of tumor cells is mediated by cytotoxic lymphocytes, cytokines, and nitric oxide.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is a potent inducer of CTL activity and cytokine production in vivo. Protein A (PA) of Staphylococcal aureus has been found to have diverse biological response modifying properties and to possess antitumor, antitoxic and antiparasitic effects. In this study we examined the anti-tumor effect of these two superantigens used separately as well as in combination in mice carrying the Ehrlich ascites tumor. With combined treatment, DNA cell cycle analysis of tumor cells showed a significant (P < 0.05) percentage of tumor cell death. Levels of the soluble mediators TNF-alpha, IFN-gamma and IL-1 as well as NO were elevated. Additionally, CD4(+) and CD8(+) specific T cells in spleen, thymus and PBMC in tumor carrying mice were increased (P < 0.01). Our data altogether suggests that enhanced tumor cell death is caused by the increased CTL activity, cytokine and nitric oxide levels, in response to the combined effect of SEA + PA.     

葡萄球菌B型肠毒素突变体的分子设计、高效表达与活性初步研究

葡萄球菌B型肠毒素（SEB0是重要的超抗原,依据SEB分子的晶体结构并结合表面分析,模拟设计了与MHCⅡ类分子和TCR VB区亲合力降低的三位点突变体mSEB(Y89A,C93S,Y94A)。通过PCR重叠延伸法获得突变体基因,导入PBV220载体中,于E.coliDH5α中获得高效表达,纯化的突变毒素T细胞激活活性仅为野生型毒素的1/5左右。     

Functional analysis of the disulphide loop mutant of staphylococcal enterotoxin C2

The superantigen staphylococcal enterotoxin C2 (SEC2) tremendously activate T lymphocytes bearing certain T-cell receptor Vβ domains when binding to MHC II molecules, which launches a powerful response of tumour inhibition in vitro as well as in vivo. However, the toxicity of SEC2 performed in clinic limited its broad application for immunotherapy. The previous studies suggested that the disulphide loop may be important for the toxicity of some SEs, which prompted us to investigate the potential roles of the disulphide loop in biological activity of SEC2. Site-directed mutagenesis was used to disturb the formation of the disulphide bond by substituting Ala or Ser for Cys-93 and Cys-110. The expressed mutants in Escherichia coli were used to determine their superantigen activity and toxicity. Results showed that all of the mutated proteins exhibited reduced abilities to induce T-cell proliferation and cytotoxic effects on tumour cells L929 and Hepa1-6, suggesting that the disulphide loop plays functional role in maintaining the maximal superantigen activity of SEC2. Furthermore, the toxicity assays in vivo showed that all of the mutants induced a reduced emetic and pyrogenic responses compared with native SEC2, which might be important for further construction of lowly toxic superantigen agent.     

Geraniin inhibits cell growth and promoted autophagy-mediated cell death in the nasopharyngeal cancer C666-1 cells

BackgroundNasopharyngeal carcinoma (NPC) is a rare malignant tumor developing from epithelial linings of nasopharynx, and 10–50 out of 100,000 NPC cases were recorded globally particularly in the Asian countries.MethodologyThe cytotoxicity of geraniin against the NPC C666-1 cells were analyzed using MTT assay. The influences of geraniin on the C666-1 cell viability with the presence of ROS and apoptosis inhibitors were also studied. The expressions of PI3K, Akt, mTOR, and autophagic markers LC3, ATG7, P62/SQSTM1 expressions in the C666-1 cells were studied by western blotting analysis. The ROS production was assayed using DCFH-DA staining. The immunofluorescence assay was performed to detect the NF-κB and β-catenin expressions in the C666-1 cells.ResultsThe cell viability of C666-1 cells were appreciably prevented by the geraniin. The geraniin treatment also inhibited the C666-1 cell growth with the presence of apoptotic inhibitor Z-VAD-FMK. The geraniin-treatment effectively improved the ROS production and inhibited the NF-κB and β-catenin expressions in the C666-1 cells. Geraniin appreciably modulated the PI3K/Akt/mTOR signaling axis and improved the autophagy-mediated cell death via improving the autophagic markers LC3 and ATG7 expressions in the C666-1 cells.ConclusionIn conclusion, our results proved that geraniin inhibits C666-1 cell growth and initiated autophagy-mediated cell death via modulating PI3K/Akt/mTOR cascade and improving LC3 and ATG7 expressions in the C666-1. Geraniin and it could be a hopeful and efficient candidate to treat the human NPC in the future.     

Differences in the expression of the human interferon-gamma gene in fresh lymphocytes and cultured lymphoblasts

Mammalian erythrocytes have been shown to bind 125I labeled ceruloplasmin. Binding was reversible and specific. Scatchard analysis yielded linear plots with a Kd of approximately 5nM. The binding site appeared to be a protein located on the cell surface. A ceruloplasmin binding protein with a molecular weight of 60,000 daltons was isolated from human erythrocytes. Erythrocytes which were not protected by ceruloplasmin's antioxidant properties, did not bind ceruloplasmin. Our results provide evidence for the presence of ceruloplasmin receptors in the erythrocyte membrane. It is proposed that the antioxidant activity of ceruloplasmin may play a role in determining the lifespan of circulating red cells.     

CYP27A1 inhibits bladder cancer cells proliferation by regulating cholesterol homeostasis

CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis, converts cholesterol into 27-hydroxycholesterol (27-HC). The relationship between CYP27A1 and cell proliferation was studied to determine the role of CYP27A1 in bladder cancer. The expression of CYP27A1 in three bladder cancer cell lines (T24, UM-UC-3 and 5637) were assessed by qRT-PCR and Western blotting, and cells with stable CYP27A1 expression were generated by lentiviral infection. Cell proliferation was detected by MTT assays, colony formation assays and a tumor xenograft model in vitro and in vivo, and the intracellular 27-HC and cholesterol secretion levels were detected by enzyme-linked immunosorbent assays (ELISA). The results revealed that CYP27A1 expression was downregulated in androgen receptor (AR)-positive T24/UM-UC-3 cells compared with AR-negative 5637 cell. After CYP27A1 expression was restored, cell proliferation was inhibited in vitro and in vivo because much more intracellular 27-HC was produced in the CYP27A1-overexpressing cells than in the control cells. Both T24 and UM-UC-3 cells treated with 27-HC showed similar results. In addition, CYP27A1/27HC could reduce the cellular cholesterol level in both T24 and UM-UC-3 cells by upregulating ATP-binding cassette transporters G1 and A1 (ABCG1 and ABCA1) through Liver X receptors (LXRs) pathway and downregulating low-density lipoprotein receptor (LDLR) expression. These findings all suggest that CYP27A1 is a critical cholesterol sensor in bladder cancer cells that may contribute significantly to bladder cancer proliferation.     

Cancer stem cells as a therapeutic target in bladder cancer

Bladder cancer is one of the most prevalent genitourinary cancers responsible for about 150,000 deaths per year worldwide. Currently, several treatments, such as endoscopic and open surgery, appended by local or systemic immunotherapy, chemotherapy, and radiotherapy are used to treat this malignancy. However, the differences in treatment outcome among patients suffering from bladder cancer are considered as one of the important challenges. In recent years, cancer stem cells, representing a population of undifferentiated cells with stem-cell like properties, have been eyed as a major culprit for the high recurrence rate in superficial papillary bladder cancer. Cancer stem cells have been reported to be resistant to conventional treatments, such as chemotherapy, radiation, and immunotherapy, which induce selective pressure on tumoral populations resulting in selection and growth of the resistant cells. Therefore, targeting the therapeutic aspects of cancer stem cells in bladder cancer may be promising. In this study, we briefly discuss the biology of bladder cancer and then address the possible relationship between molecular biology of bladder cancer and cancer stem cells. Subsequently, the mechanisms of resistance applied by cancer stem cells against the conventional therapeutic tools, especially chemotherapy, are discussed. Moreover, by emphasizing the biomarkers described for cancer stem cells in bladder cancer, we have provided, described, and proposed targets on cancer stem cells for therapeutic interventions and, finally, reviewed some immunotargeting strategies against bladder cancer stem cells.     

超抗原葡萄球菌肠毒素A、B和C1的T细胞抗原HLA表位预测

为预测超抗原葡萄球菌肠毒素（SE）家族中的SEA、SEB和SEC1的HLAⅠ和HLAⅡ抗原结合表位,并对其活化T细胞作用的机理进行探讨,根据已发表的SEA、SEB和SEC1基因全序列,用T细胞抗原表位预测软件Guotif2.0对其进行T细胞抗原表位预测,统计与HLAⅠ和HLAⅡ各抗原位点结合的SEA、SEB和SEC1肽段的出现次数。结果显示,SEA、SEBT SEC1具有共同的特点,即都是主要与HLAⅠ类分子的A3位点和HLAⅡ类分子的DR1位点具有较强的结合。说明SEA、SEB和SEC1与HLAⅠ类分子和HLAⅡ类分子都有很强的结合性。三者在HLAⅠ和HLAⅡ结合位点上具有较强的同源性。本研究为SE活化T细胞作用机制的功能实验提供了依据。     

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins

Summary The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.     

Inhibition of NEDD4 inhibits cell growth and invasion and induces cell apoptosis in bladder cancer cells

The neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) plays a pivotal oncogenic role in various types of human cancers. However, the function of NEDD4 in bladder cancer has not been fully investigated. In the present study, we aim to explore whether NEDD4 governs cell proliferation, apoptosis, migration, and invasion in bladder cancer cells. Our results showed that downregulation of NEDD4 suppressed cell proliferation in bladder cancer cells. Moreover, we found that inhibition of NEDD4 significantly induced cell apoptosis. Furthermore, downregulation of NEDD4 retarded cell migration and invasion. Notably, overexpression of NEDD4 enhanced cell growth and inhibited apoptosis. Consistently, upregulation of NEDD4 promoted cell migration and invasion in bladder cancer cells. Mechanically, our Western blotting results revealed that downregulation of NEDD4 activated PTEN and inhibited Notch-1 expression, whereas upregulation of NEDD4 reduced PTEN level and increased Notch-1 level in bladder cancer cells. Our findings indicated that NEDD4 exerts its oncogenic function partly due to regulation of PTEN and Notch-1 in bladder cancer cells. These results further revealed that targeting NEDD4 could be a useful approach for the treatment of bladder cancer.     

哮喘患者外周血单核细胞血红素氧合酶-1表达水平的研究

目的探讨血红素氧合酶-1(HO-1)在哮喘患者外周血单核细胞(PBMC)的表达及与肺通气功能的关系.方法应用免疫组织化学和逆转录聚合酶链反应技术分析18例哮喘患者PBMC的 HO-1蛋白及mRNA水平的表达,测定全血一氧化碳血红蛋白(COHb)的百分比含量、血清总IgE含量、肺通气功能,并与18名健康正常者的结果进行比较.结果哮喘组PBMC中HO-1表达阳性的细胞百分比41.7%±7.44%与正常对照组10.5%±4.36%比较,差异有显著性(P<0.01),哮喘组PBMC HO-1 mRNA表达的平均吸光度值(26.05±4.14)与正常对照组(10.82±4.26)比较,差异亦有显著性(P<0.01),HO-1染色阳性细胞的百分比与FEV1占预计值%、PEFR和MEFR50%均呈显著负相关(r值分别为-0.89,-0.56,-0.51,均P<0.01),与全血COHb的百分比含量及血清总IgE含量呈显著正相关(r值分别为0.80, 0.48, 均P<0.05).HO-1 mRNA的表达水平与1s用力呼气容积(FEV1)占预计值%、顶峰呼气流速(PEFR)和50%肺活量时的最大呼气流速(MEFR50%)均呈显著负相关(r 值分别为-0.89,-0.65,-0.67, 均P<0.01),与全血COHb的百分比含量和血清总IgE含量呈显著正相关(r分别为 0.85和0.62, 均P＜0.01).结论哮喘患者PBMC 的HO-1表达水平显著增加,提示HO-1可能参与了哮喘的发病过程,HO-1表达的变化与哮喘患者的病情程度有一定关系.     

Biological analysis of the deletion mutants of Staphylococcal enterotoxin C2

To investigate the functional domains involved in the biological activity of staphylococcal enterotoxin (SEC2), a series of SEC2 mutants were constructed. Deletion of the last 77 amino acids at the C-terminus of SEC2 did not affect its native superantigen and fever activities, and further removal of the C-terminal residues reduced SEC2 activities significantly. On the other hand, the mutants lacking 18 or more N-terminal residues severely impaired superantigen activity. These data indicated that the functional regions for the biological activities of SEC2 were confined to N-terminal domain, further implied that the proper three-dimensional structure of SEC2 is not needed for its biological activities. Our results deliver valuable information that it is possible to design new SEC2 immunotherapeutic agents which have the superantigen activity and low molecular weight for permeability.     

Cytokines Released by Human Peripheral Blood Mononuclear Cells Inhibit the Production of Early and Late Cytomegalovirus Proteins

Cytomegalovirus-infected human fibroblasts are susceptible to lysis by natural killer cells and cytotoxic T cells. The purpose of this study was to determine whether non-lytic mechanisms might also contribute to the control of cytomegalovirus infection. The appearance of cytomegalovirus proteins in infected fibroblasts was determined by flow cytometry. Infected fibroblasts incubated with peripheral blood mononuclear cells for 3 days expressed less early and late proteins than fibroblasts incubated without peripheral blood mononuclear cells. Supernatants generated by the cocultivation of peripheral blood mononuclear cells with cytomegalovirus-infected fibroblasts inhibited the production of cytomegalovirus early and late proteins. The soluble factors in supernatants which contributed to the inhibitory effect were identified as interferons α, β and γ, and tumor necrosis factors α and β. The ability of supernatants to inhibit the production of cytomegalovirus early protein was mimicked by combinations of corresponding recombinant cytokines. The inhibition of cytomegalovirus protein production by cytokines produced by peripheral blood mononuclear cells may contribute to early containment of cytomegalovirus infection.     

Inhibition of stearoyl CoA desaturase‐1 activity suppresses tumour progression and improves prognosis in human bladder cancer

Urinary bladder neoplasm is one of the most common cancers worldwide. Cancer stem cells (CSCs) have been proven to be an important cause of cancer progression and poor prognosis. In the present study, we established bladder CSCs and identified the crucial differentially expressed genes (DEGs) between these cells and parental bladder cancer cells. Analyses of bioinformatics data and clinical samples from local hospitals showed that stearoyl CoA desaturase‐1 (SCD) was the key factor among the DEGs. A significant correlation between SCD gene expression and poor prognosis among patients with bladder cancer was observed in our data. Loss‐of‐function experiments further revealed that the SCD inhibitor A939572 and SCD gene interference reduced cell proliferation and invasion. The above data suggest that SCD may serve as a novel marker for the prediction of tumour progression and poor prognosis in patients with bladder cancer.