This study was aimed to investigate the effect of clostridium difficile toxin A (Tcd A) on proliferation of K562 cells and its mechanism. The proliferative activity of K562 cells exposed to Tcd A was tested by MTT assay; cell cycle distribution and mitochondrial membrane potential were analyzed by flow cytometry; the protein expression of cytochrome C and DNA fragmentation were observed by immunohistochemistry staining and agarose gel electrophoresis respectively. The results indicated that Tcd A inhibited proliferation of K562 cells in a time-and concentration-dependent manner. Cells were arrested at G(0)/G(1) phase. Peak of apoptosis appeared. The protein expression of cytochrome C increased as compared with control group (p < 0.05). Agarose gel electrophoresis of DNA from K562 treated with Tcd A revealed a "ladder" pattern. It is concluded that clostridium difficile toxin A can inhibit proliferation and induce apoptosis of K562 cells. The mechanism may be in relation to decrease of mitochondrial membrane potential and the release of cytochrome C from mitochondria matrix.
Apoptosis ; drug effects ; Bacterial Toxins ; pharmacology ; Cell Proliferation ; drug effects ; Enterotoxins ; pharmacology ; Humans ; K562 Cells

OBJECTIVEMouse factor VII (mfVII), ligand of tissue factor (TF) which is frequently over-expressed during neovascularization activated by tumor growth, was fused to staphylococcus enterotoxin A (SEA) that mediates greater intensity of T-cell activation against tumor cells. The anti-tumor effects of the mfVII-SEA chimeric protein were evaluated.

METHODSFusion of SEA and mfVII cDNA was constructed using adenovirus vector and produced in 293 packaging cell lines. The 293 cells containing the adenovirus were administered subcutaneously to mice. Fluorescence studies at the injection site and the liver were performed 3 days later. Mouse prostatic tumor RM-1 cells and mouse sarcoma MCA 205 H12 cell lines were then used in mice to create lung metastasis and subcutaneous tumor to carry out efficacy evaluation, respectively.

RESULTSAdenovirus released from the injected 293 cells only infected the subcutaneous tissue at the injection site. The in vivo experiments in mice revealed that formation of lung metastasis was strongly inhibited by the mfVII-SEA (23 +/- 8) compared to the vacant vector control group (193 +/- 38) and PBS control group (211 +/- 42) (P < 0.01). The mfVII-SEA also strongly suppressed tumor growth at the subcutaneous injection site (342.6 +/- 107.1) mm(3) compared to that of vacant vector control (2244.3 +/- 350) mm(3) and SEA (1208.3 +/- 210) mm(3) by the 23rd day.

CONCLUSIONThe chimeric protein mfVII-SEA significantly inhibits lung metastasis formation and local tumor growth.

Animals ; Antigens, Bacterial ; genetics ; immunology ; pharmacology ; Antineoplastic Agents ; pharmacology ; Enterotoxins ; genetics ; immunology ; pharmacology ; Factor VII ; genetics ; pharmacology ; Female ; Lung Neoplasms ; secondary ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Prostatic Neoplasms ; pathology ; Recombinant Fusion Proteins ; genetics ; pharmacology ; Staphylococcus ; Thromboplastin ; genetics ; pharmacology

OBJECTIVETo investigate the contamination of Staphylococcus aureus isolates in ice cream by phenotypic typing and molecular typing.

METHODSThe Staphylococcus aureus isolates were separated from ice cream, filler, cutter, salves and material. The separated isolates were characterized by drug-resistance, staphylococcal enterotoxin (SEA-E), SE (A-E, G-J) genes and pulsed-field gel electrophoresis (PFGE) types.

RESULTTwo Staphylococcus aureus isolates were separated, one from ice cream, another from cutter. Their characteristics of drug-resistance, staphylococcal enterotoxin (SEA-E), SE (A-E,G-J) genes and PFGE type were the same.

CONCLUSIONThe two Staphylococcus aureus isolates were the same clone. The contaminated Staphylococcus aureus isolates could be traced to the contaminated cutters.

Anti-Bacterial Agents ; pharmacology ; Bacterial Typing Techniques ; Electrophoresis, Gel, Pulsed-Field ; Enterotoxins ; genetics ; Food Microbiology ; Ice Cream ; microbiology ; Microbial Sensitivity Tests ; Staphylococcus aureus ; classification ; drug effects ; isolation & purification

The anti-tumor effect and mechanism of the staphylococcal enterotoxin A (SEA) were studied. The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumor cells (MGC80-3). The experimental group was treated with SEA, and the control group was treated with normal saline. The percentage of tumor generation and tumor mass was measured. The results showed that the percentage of the tumor generation in the SEA-treated mice was lower than in the control group, but there was no significant difference (P > 0.05). However, the tumor mass in the experimental group was significantly lighter than in the control group, with the difference being very significant (P < 0.001). There were more CD4+ T cells and CD8+ T cells in the tumor of the mice treated with SEA than those of the control group. SEA has an obvious anti-tumor effect on mice gastric tumor. The mechanism might be that SEA induces the effect of superantigen-dependent cell mediated cytotoxicity to the tumor cells.
Adjuvants, Immunologic ; pharmacology ; Animals ; Antineoplastic Agents ; pharmacology ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Enterotoxins ; immunology ; pharmacology ; Female ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Staphylococcus aureus ; immunology ; Stomach Neoplasms ; immunology ; pathology ; Superantigens ; immunology ; pharmacology

OBJECTIVETo clone the LTB gene of E.coli and the CTB gene of V.cholerae, and to construct expression vectors of these genes.

METHODSThe LTB gene from E.coli strain 44815 and the CTB gene from V.cholerae strain eastern 74 were amplified by high fidelity PCR. The nucleotide sequences of the two target DNA amplification fragments were sequenced after T-A cloning. pET32a expression vectors with inserted LTB and CTB genes were constructed. The LTB and CTB fusion proteins were expressed in E.coli strain BL21DE3 inducted by IPTG at different dosages. The two expression products were identified by SDS-PAGE and G(M1)-ELISA.

RESULTSIn comparison with the reported LTB and CTB sequences, the nucleotide sequence homologies of the cloned LTB gene and CTB gene were from 99.12% approximate, equals 99.71% and 98.54% approximate, equals 99.42%, while their putative amino acid sequence homologies were as high as 97.58% approximate, equals 99.19% and 96.77% approximate, equals 99.19%. The expression outputs of LTB and CTB fusion proteins in pET32a LTB BL21DE3 and pET32a-CTB-BL21DE3 systems were approximately 30% and 10% of the total bacterial proteins, respectively. The LTB and CTB fusion proteins were able to combine with bovine G(M1) confirmed by ELISA.

CONCLUSIONThe expression systems of LTB and CTB genes have been successfully established. Both the expressed LTB and CTB fusion proteins possess mucosal adjuvant immunoactivity.

Adjuvants, Immunologic ; pharmacology ; Animals ; Bacterial Toxins ; genetics ; pharmacology ; Base Sequence ; Cholera Toxin ; genetics ; pharmacology ; Cloning, Molecular ; Enterotoxins ; genetics ; pharmacology ; Escherichia coli ; genetics ; Escherichia coli Proteins ; Immunity, Mucosal ; Rabbits ; Vibrio cholerae ; genetics

It has been well established that bacterial superantigens lead to the induction and aggravation of chronic inflammatory skin diseases. We investigated the clinical significance of serum specific immunoglobulin E (lgE) to the staphylococcal superantigens staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), and toxic shock syndrome toxin (TSST)-1 in patients with chronic urticaria (CU), focusing on the differences in these prevalences between aspirin-intolerant CU (AICU) and aspirin-tolerant CU (ATCU) patients. Aspirin sensitivity was confirmed by oral aspirin provocation test. There were 66 patients AICU and 117 patients ATCU in the study. Serum IgE antibodies specific for SEA, SEB, and TSST-1 were measured by the ImmunoCAP test and the patients were compared with 93 normal controls (NC). The prevalences of serum specific IgE to staphylococcal superantigens were significantly higher in CU than in NC patients (IgE to SEA, 13.7% vs. 5.4%; IgE to SEB, 12.0% vs. 4.3%; IgE to TSST-1, 18.0% vs. 6.5%; p<0.05, respectively). The patients with specific IgE to SEA, SEB, and TSST-1 had higher serum total IgE levels and higher rates of atopy. Significant associations were noted between the prevalence of specific IgE to SEA and SEB and the HLA DQB1*0609 and DRB1*1302 alleles in the AICU group. We confirmed that a sub-population of patients with CU possesses serum IgE antibodies to SEA, SEB, and TSST- 1. Particularly, the IgE immune response to TSST-1 is associated with aspirin sensitivity in CU patients.
Adolescent ; Adult ; Aged ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Antigens/*chemistry ; Aspirin/pharmacology ; Bacterial Toxins/metabolism ; Chronic Disease ; Enterotoxins/metabolism ; Female ; Humans ; Immunoglobulin E/*chemistry/metabolism ; Male ; Middle Aged ; Phenotype ; Staphylococcus/*genetics/immunology ; Superantigens/metabolism ; Urticaria/*immunology

This study was purposed to investigate the growth inhibition and apoptosis-inducing effect of Clostridium difficile toxin A (TcdA) on the leukemia cell line K562. The proliferative activity of K562 cells exposed to Tcd A was tested by MTT assay, cell apoptosis was detected by flow cytometry; immunocytochemistry and colorimetric assay were employed to detect the protein expressions of BCL-2/BAX and the activity of Caspase-3, respectively. The results indicated that the proliferation of K562 cells was inhibited in a time-and dose-dependent manner after exposure to Tcd A for 24, 48 and 72 hours, the cells displayed the typical apoptotic, morphological changes, the expression of BCL-2 protein was down-regulated but the expression of BAX protein was signficantly increased, compared with control group (p < 0.05). In addition, caspase-3 was activated in a concentration-dependent manner. It is concluded that Tcd A inhibits cell growth of K562 by inducing apoptosis, and the up-regulation of BAX protein and activation of caspase-3 may play important roles in these processes.
Apoptosis ; drug effects ; Bacterial Toxins ; pharmacology ; Caspase 3 ; metabolism ; Enterotoxins ; pharmacology ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo evaluate the effects of Staphylococcus aureus enterotoxin B (SEB) on proinflammatory cytokine/chemokine releases in primary human nasal epithelial cell (HNEC).

METHODSEpithelial cells of nasal polyps (NP) and inferior turbinate (IT) were cultured without serum under stimulus of SEB 1, 10, 100 ng/ml, IL-1beta 20 ng/ml and SEB 10 ng/ml + dexamethasone 13 ng/ml for 12,24 and 48 h, respectively. The expression of IL-5 and Granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA derived from epithelial cells was detected by in situ hybridization.

RESULTS(1) The expression of IL-5 and GM-CSF mRNA was time and dose-dependent, and reached to a peak under SEB 10 ng/ml for 24 h (P < 0.05). The mRNA expressed more intensively in epithelial cells from NP than IT (P < 0.05). (2) The expression of IL-5 and GM-CSF mRNA increased less under the stimulus of IL-1beta than SEB 10 ng/ml (P < 0.05). (3) The mRNA level of IL-5 and GM-CSF decreased under the stimulus of SEB + dexamethasone 13 ng/m when compared with the stimulus of SEB 10 ng/ml (P < 0.05).

CONCLUSIONSEB showed proinflammatory effects on HNEC.

Cells, Cultured ; Dexamethasone ; pharmacology ; Enterotoxins ; pharmacology ; Epithelial Cells ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Humans ; Interleukin-1beta ; metabolism ; Interleukin-5 ; metabolism ; Nasal Mucosa ; cytology ; metabolism ; pathology ; RNA, Messenger ; metabolism

The rare codons of a fragment in staphylococcal enterotoxin A gene were turned into the most high usage frequency codons in E. coli by overlap PCR technique. Genes of sea and seam were cloned into 7ZTS expression vector and transformed into JM109(DE3), respectively. The result shows that expression level of sea gene was very low, but the expression level of seam was as high as 15% of total cell proteins. The expression product shows activity of antitumor in vivo.
Animals ; Base Sequence ; Codon ; genetics ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins ; genetics ; metabolism ; pharmacology ; Escherichia coli ; genetics ; Gene Expression Regulation, Bacterial ; Male ; Mice ; Molecular Sequence Data ; Neoplasms, Experimental ; drug therapy ; pathology ; Point Mutation ; Recombinant Proteins ; genetics ; metabolism ; pharmacology

BACKGROUND AND OBJECTIVEImmunocompromised patients with malignant tumor always lack of strong anti-tumor immune response, because the antigenicity of tumor cells is weak, and antigen-presenting cell function is low, so that can not be effectively presenting tumor antigens to the lymphocytes. Therefore, how to effectively induce anti-tumor immune response is the key issue. Through the study on establishing a method to culture dendritic cells (DC) in vitro and to observe the anti-lung cancer immunological effect induced by DC, we provided definite experiment basis for the clinic application of vaccine based on DC.

METHODSThrough the experiment we get the soluble antigen polypeptide from lung cancer cells GLC-82 by 3 mol/L potassium chloride. DCs are cultured and obtained from peripheral blood mononuclear cell by GM-CSF, IL-4 and TNF-a. DCs are identified by flow cytometer (FCM) and immunostaining. DCs modified by lung cancer tumor soluble antigen (TSA) and staphylococcal enterotox in A (SEA), DCs modified by TSA or DCs modified by SEA or DCs modified by nothing were cultivated together with T lymphocyte, and the obtained cells are named TSA-SEA-DCL or TSA-DCL or SEA-DCL or DCL as effector cells. The anti-tumor activity of every effector cells against target cells was assayed with MT method. Shape of DCs and effector cells, and the process of killing target cells were observed in microscope.

RESULTSInduced DCs expressed more CD1a, CD80 and HLA-DR, which had typical cell traits such as tree branch. The killing ratio of the TSA-SEA-DCL in vitro to GLC-82 is larger than TSA-DCL, SEA-DCL and DCL, also larger than to K562. When the effector cells cultivate with target cells, we can observe the CTL approach and gather to the cancer cell, induce it necrosis and apoptosis.

CONCLUSIONRipe DCs that have typical characteristic and phenotype could be induced successfully. High potency and relatively specific antilung caner effect can be prepared in virtue ofDC Bacterin Induced by lung caner TSA and SEA.

Antigens, Neoplasm ; immunology ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; Enterotoxins ; immunology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunotherapy ; Interleukin-4 ; pharmacology ; Lung Neoplasms ; immunology ; therapy ; Superantigens ; immunology ; T-Lymphocytes, Cytotoxic ; immunology