Chimeric specific antigen epitope-carrying dendritic cells induce interleukin-17(+) regulatory T cells to suppress food allergy.

BACKGROUND: The on-purpose-modulated dendritic cells (DCs) have shown charming effects on restoring immune regulatory functions in subjects with immune diseases. OBJECTIVE: This study aims to construct DCs carrying chimerical antigen (Ag) peptides (CAP-DCs) to induce interleukin (IL)-17+ inducible Tregs (iTregs) to alleviate food allergy (FA) in a murine model. METHODS: In this study, we constructed CAP-DCs. The CAP is a fusion protein, consisting of a segment of recombinant scFv of anti-DEC205 antibody and an ovalbumin (OVA) epitope (IC). A murine OVA-FA model was developed to test the effects of CAP-DCs on suppressing the allergic response in the intestine. RESULTS: The CAP-DCs are characterized as that a complex of scFv-IC is presented on the surface of the cells, moderately express CD80 and CD86 as well as IL-6, IL-23, transforming growth factor (TGF)-ß and CCR9. After being passively transferred with CAP-DCs or injection of scFv-IC, Ag-specific IL-17+ Foxp3+ iTregs were induced in the intestinal lamina propria of FA mice. The iTregs showed immune suppressive effects on Ag-specific Th2 response. FA mice were adoptively transferred with the CAP-DCs or scFv-IC injection, which resulted in a significant decrease in the number of Ag-specific Th2 cells and suppression of FA response in an Ag-specific manner. CONCLUSIONS AND CLINICAL RELEVANCE: CAP-DCs can ameliorate FA response by inducing Ag-specific IL-17+ Foxp3+ iTregs and suppressing Ag-specific Th2 response. To generate CAP-DCs has the translational potential in the treatment of FA.

[The expression vector of murine secreting IL-1ß promotes proliferation and migration of Hepa1-6 hepatoma cells].

AIM: To establish a murine secreted mature peptide IL-1ß expression vector, transfect into Hepa1-6 hepatoma cells, and analyze the effect of recombinant IL-1ß on proliferation, migration, and its specific expression in Hepa1-6 hepatoma cells. METHODS: The murine AFP signal peptide encoding sequence and mature IL-1ß encoding fragment were linked together through overlapping PCR, and the chimeric DNA sequence was then inserted into a liver specific expression vector pLIVE(TM); to make a recombinant pLIVE-smIL-1ß which expressed secreted murine IL-1ß of classical pathway. pLIVE-smIL-1ß, pLIVE(TM); and pLIVE-lacZ were transfected into Hepa1-6 by jetPEI respectively. Transfection of the vectors were detected by ß-gal staining using pLIVE-lacZ transfectants. Cells treated with 5 µg/mL lipopolysaccharide were used as positive control and 3 µmol/L monesin was added into culture system to block classical pathway secretion, then sandwich ELISA was employed to detect the IL-1ß levels both in supernatant and in cytoplasm of each group of transfected cells. The proliferation of Hepa1-6 hepatoma cells was determined by MTT assay and migration of Hepa1-6 cells was assessed by scratch test in vitro. RESULTS: pLIVE-smIL-1ß vector successfully expressed murine IL-1ß in Hepa1-6 cells. Expression of the recombinant vector peaked at day 3 as indicated in a ß-gal staining method. After transfection, compared with Hepa1-6/mock cells, IL-1ß expression levels both in supernatant and in cytoplasm of Hepa1-6/smIL-1ß cell were significantly increased detected by ELISA. The proliferation of Hepa1-6/smIL-1ß group was markedly promoted in vitro detected by MTT assay. CONCLUSION: The recombinant expression vector can secret IL-1ß through classical pathway which significantly promoted proliferation and migration of hepatoma cells in vitro.

[Preparation and characterization of monoclonal antibodies for human BCSC-1 protein].

AIM: To obtain monoclonal antibodies against human BCSC-1 protein for further study of the structure and function of human BCSC-1 protein. METHODS: pET-30a-BCSC-1 plasmid was constructed and transformed into E.coli BL21 (DE3) to express recombinant protein. BALB/c mice were immunized with recombinant human BCSC-1 protein, hybridoma cell lines secreting monoclonal antibodies against human BCSC-1protein were screened by regular cell fusion and subcloning approach. The specificity of these monoclonal antibodies were determined by ELISA and Western blotting. Expression of BCSC-1 protein in pcDNA3.1/v5-HisB-BCSC-1transfected MCF-7 cells was identified by Immunohistochemistry. RESULTS: Successfully constructed a prokaryotic expression vector pET30a-BCSC-1.BCSC-1 protein was expressed in E.coli BL21 (DE3). One hybridoma cell line 1B3 stable in secreting specific monoclonal antibodies wa successfully obtained. It could bind specifically to BCSC-1 protein proved by Western blotting and Immunohistochemistry assay. CONCLUSION: Monoclonal antibodies of high specificity against human BCSC-1 protein has been successfully prepared, which laid the foundation for the further study of human BCSC-1 protein.

[Human hnRNP I prokaryotic expression and preliminary application.].

AIM: To express recombinant protein hnRNP I with prokaryotic expression system and assess the presence of autoantibodies against hnRNP I in systemic sclerosis (SSc) as well as other CTD. METHODS: Human hnRNP I gene was amplified by RT-PCR from HeLa cells and cloned into pET-30a vector , then pET-30a-hnRNP I plasmid was transferred into E.coli BL21 (DE3) to express recombinant protein. The sera from patients including SSc, SLE, SS, MCTD, UCTD, RA and controls were detected by ELISA with the purified recombinant hnRNP I protein. RESULTS: The recombinant protein was highly expressed in E.coli BL21(DE3) and specially reacted with clinically diagnosed SSc patients's serum. The result suggested that the autoantibodies against hnRNP I had higher positive ratio(48.72%) in SSc than other serum of AID and control. CONCLUSION: Human hnRNP I protein was successfully expressed in prokaryotic expression system. The purified hnRNP I protein can be used to diagnosis of SSc.

Inhibition of alpha-mannosidase Man2c1 gene expression suppresses growth of esophageal carcinoma cells through mitotic arrest and apoptosis.

To study the effects of suppressed alpha-mannosidase Man2c1 gene expression on EC9706 human esophageal carcinoma cells, the cells were treated with short interfering RNA. Growth inhibition of EC9706 cells was observed when Man2c1 expression was inhibited in this way. Flow cytometric analysis showed accumulation of cells in S and G(2)-M phases, as well as cell apoptosis. The mitotic index test showed cell-cycle arrest at the M checkpoint. Although the percentage of cells in (pro)metaphase increased, the proportion of cells in anaphase and telophase decreased. Apoptosis was trigged by mitotic arrest. Furthermore, microtubules in EC9607 cells were examined by means of fluorescence staining of alpha-tubulin. Although control cells showed a nest-like microtubule network, the microtubule network in experimental cells was vague and condensed at the perinuclear region. Some cells with Man2c1 suppression had large protrusions of cytoplasm, some of which linked with the main body through a long, thin connection. Western blotting showed that tubulin polymerization was inhibited. The data imply that induction of mitotic arrest and consequent apoptosis resulted from microtubule disorganization, which appears to be one of the major cellular mechanisms by which suppressed expression of the Man2c1 gene causes growth inhibition of EC9706 esophageal carcinoma cells. In addition, Man2c1 suppression results in upregulation of E-cadherin, alpha-catenin, and beta-catenin expression in cells.

[Experimental BCSC-1 gene therapy on nasopharyngeal carcinoma mediated by adenovirus].

OBJECTIVE: To explore the therapeutic value of BCSC-1 in tumor gene therapy. METHODS: Recombinant adenovirus Ad5-BCSC-1 was prepared. Cell proliferation was assayed using CellTiter 96 Aqueous one solution cell proliferation assay kit. Ad5-BCSC-1 was injected into tumor with Ad5-egfp or with PBS injection as controls. The injections were repeated one weak later. After another 2 weeks, the mice were sacrificed and the tumors were excised and weighed. RESULTS: The growth of the CNE-2L2 cell infected with Ad5-BCSC-1 in vitro was remarkably slower than that of the controls, the wild type cell and the cell infected with Ad5-egfp. In contrast to the controls, the cells infected with Ad5-BCSC-1 aggregated and formed huge messes in the culture. The average weight of the CNE-2L2 tumors in mice was (2.28 +/- 0.73), (2.07 +/- 0.40), and (0.58 +/- 0.32) g for the tumors injected with PBS, Ad5-egfp, and Ad5-BCSC-1, respectively (Ad5-BCSC-1 vs. PBS or Ad5-egfp, P<0.05). CONCLUSION: Intra-tumor injection of Ad5-BCSC-1 can suppress the growth of CNE-2L2 tumor in nude mice, suggesting that BCSC-1 gene therapy may be effective for tumors with low or no expression of BCSC-1 gene.

[Inhibition of malignant activities of nasopharyngeal carcinoma cell by ectopic expression of BCSC-1 gene].

OBJECTIVE: To study effects of ectopic expression of BCSC-1 gene on the malignant activi-BCSC-1 cDNA was isolated by RT-PCR ties of human nasopharyngeal carcinoma cell CNE-2L2. METHODS: and inserted into pMAL-c2X and pcDNA4/myc-His A. BCSC-1 protein was expressed in prokaryocytes. Rabbit antiserum to BCSC-1 was developed by means of immunization of rabbit with the BCSC-1 protein. Expression of BCSC-1 gene in wild type CNE-2L2 cell (W cell) was examined by real-time RT-PCR and immunofluorescence staining with the antiserum as a probe. pcDNA4/myc-His A-BCSC-1 was transfected into W cell at the presence of LipofectAmine. The cells were selected by G418 and cloned. Ectopic expression of BCSC-1 gene in W cell was examined by Western blot. Cell growth was detected by drawing of growth curves and colony formation tests. Cells were inoculated into nude mice. Size of tumors was assayed once a week. Lungs of the mice were sectioned continuously and metastatic loci in lungs were examined upon a microscope. RESULTS: Rabbit BCSC-1 antiserum was prepared. Expression of BCSC-1 gene in W cell was found to be very low. CNE-2L2 cell with ectopic expression of BCSC-1 gene was developed. Growth in vitro, colony formation, tumorigenesis in nude mice, and lung metastasis of the tumor were profoundly inhibited of the cell with ectopic expression of BCSC-1 gene in comparison with controls, wild type cell and the cell transfected with mock. Conclusion Ectopic expression of BCSC-1 gene exerts profound inhibitive effect on the malignant activities of CNE-2L2 cell.

[Reduction of CD44 expression results in growth inhibition of human nasopharyngeal carcinoma cell CNE-2L2 in vitro].

OBJECTIVE: To study the effect of the inhibition of CD44 gene expression on the growth of human nasopharyngeal carcinoma cell CNE-2L2 in vitro. METHODS: CD44 gene expression in cells was suppressed by siRNA which was introduced into cells through retrovirus infection. Integration of siRNA into genomic DNA was examined by genomic PCR. CD44 gene expression in cells was detected by Western blot analysis. Cell growth in vitro was assayed using Cell Titer 96 AQueous One Solution Cell Proliferation Assay kit Promega. Cells were stained with propidium iodium and cell DNA content was detected upon a flow cytometer. RESULTS: siRNA was integrated into genomic DNA of host cells. The 4 cell pools integrated with one of the 4 siCD44s showed a significant inhibition of CD44 gene expression comparing to the controls, the wild type cell and the cell pool integrated with siegfp. The cell pools integrated with siCD44-1 or siCD44-2 showed the most profound inhibition. Growth of these 2 cells in vitro was compared to that of the controls and was found to be significantly inhibited. Cell DNA content analysis indicated 44.4%, 45.5%, 53.9%, and 53.3% in G0/G1 phase; 39.3%, 40.0%, 27.1%, and 28.2% in S phase; and 16.3%, 14.5%, 19.0%, and 18.5% in G2/M phase for the wild type cell, the cell pool integrated with siegfp, the cell pools integrated with siCD44-1, and the cell pools integrated with siCD44-2, respectively. CONCLUSION: Reduction in CD44 expression inhibit the growth of CNE-2L2 cell and affects the development of cells from G0/G1 into S phase, but may somehow promote cells to develop from S into G2/M phase.

[Effects of human alpha-mannosidase Man2c1 transgene on growth and metastasis of transplanted tumor in mice].

OBJECTIVE: To study the effect of human alpha-mannosidase Man2c1 transgene on tumor growth and metastasis in mice. METHODS: Hepatoma cell H22 or squamous epithelial carcinoma cell S180 was subcutaneously inoculated into the right armpit of mice (wild type mice and 28#, 35#, and 54# transgenic mice). Tumor size was measured every week. Mice were sacrificed on day 9 or 10 and then the tumors were exercised and weighted. Tumors and lungs were fixed in formaldehyde and sectioned. The sections were stained with hematoxylin/eosin and examined under microscope. The red blood cells in spleen were destroyed by Tris-NH4Cl. Natural killer (NK) cell activity was detected with Yac-1 cell as target. RESULTS: H22 and S180 tumors grew faster in all the three transgenic mice (28#, 35#, and 54#) than in wild type mice. The average size and weight of tumors between the transgenic mice and wild type mice were significantly different (P<0.05). Most tumors in the transgenic mice invaded the surrounding tissues. In contrast, nearly all the tumors in wild type mice were capsulized. Three of 10 28# transgenic mice, 5 of 10 35# transgenic mice, 3 of 10 54# transgenic mice, and 1 of 10 wild type mice showed lung metastasis of H22 tumor. Two of 6 28# transgenic mice, 3 of 6 35# transgenic mice, 1 of 6 54# transgenic mice, and 0 of 6 wild type mice showed lung metastasis of S180 tumor. No difference of NK activity in spleen cells was observed between the transgenic mice and wild type mice. CONCLUSIONS: hMan2c1 transgene promotes growth, invasion, and metastasis of transplanted H22 and S180 tumors in mice. hMan2cl transgene does not affect NK activity in splenocytes.

[Ectopic expression of BCSC-1 gene results in enhancement of adhesion and cell cycling blockade of nasopharyngeal carcinoma CNE-2L2 cell].

OBJECTIVE: To study mechanisms of reduction of the malignant activities of human naso-pharyngeal carcinoma cell CNE-2L2 induced by ectopic expression of BCSC-1 gene. METHODS: DNA was stained with propidium iodide and assayed upon a flow cytometer. Chromosomes were stained with Hoechest 33258. Adhesion of CNE-2L2 cells was detected by cell aggregation test. Protein expression on CNE-2L2 cells was examined by Western blot. RESULTS: Cell cycle analysis showed that the percentage of CNE-2L2 cells was 55.1%, 43.4%, and 39.4% in G0/G1 phase, 25.2%, 28.7%, and 30.9% in S phase, and 19.7%, 27.9%, and 29.7% in G2/M phase for the cell with ectopic expression of BCSC-1 gene, wild type cell (W cells), and the cell transduced with the mock (M cell). Many mitotic cells were found in W cells and M cells. In contrast, almost no mitotic cell was observed in the cells with ectopic expression of BCSC-1 gene. Ectopic BCSC-1 expression resulted in cell aggregation, enhanced expression of E-cadherin, cx-catenin, and p53. CONCLUSIONS: Ectopic BCSC-1 expression causes enhancement of adhesion of CNE-2L2 cells associated with enhanced expression of E-cadherin and alpha-catenin, arrest of cell in G1 phase, which may be associated with enhanced expression of p53. These alteration may play a role in the reduction of malignant activities of the cells with ectopic expression of BCSC-1 gene.

[8B7 spectrin--a new member of spectrin family].

OBJECTIVE: To analyze the nature of the protein encoded by 8B7cDNA (1 835 bp) and to examine the localization of the protein in cells. METHODS: The nature of the protein was analyzed using Blastn, Blastp, and TMpred. Expressions of 8B7 mRNA in tissues and cells were examined by Northern blotting. Recombinant expression vectors for localization test were constructed and transfected into COS-7 cells. Fluorescence emission in cells was examined upon a laser scan confocal microscope. RESULTS: The protein encoded by 8B7cDNA was 363 amino acids long and contained spectrin repeats. It was completely homologous to the C-terminal 363 amino acids of Enaptin, Nasprin-1, Mynel, and Syne-1 proteins. It belonged to Spectrin super-family and was called 8B7 spectrin. Northern blotting revealed a 1.8 kb mRNA expression in human spleen and small intestine tissues. EGFP-8B7 fusion protein was observed to express on the nuclear membrane and in the cytoplasm in a reticular form in a localization assay on COS-7 cells. The position of fluorescence in COS-7 cells transfected with pEGFP-delta SR8B7 was similar to that in the cells transfected with pEGFP-8B7cDNA. CONCLUSIONS: 8 B7 spectrin is a novel member of spectrin super-family. It localizes to the nuclear membrane and the cytoplasm in a reticular form in COS-7 cells. The localization is determined by the C-terminal KASH domain in 8B7 spectrin molecule.

Inhibition of the alpha-mannosidase Man2c1 gene expression enhances adhesion of Jurkat cells.

Protein N-glycosylation plays very important roles in immunity and alpha-mannosidase is one of the key enzymes in N-glycosylation. This paper reports that inhibition of alpha-mannosidase Man2c1 gene expression enhances adhesion of Jurkat T cells. In comparison to the controls with normal expression of the enzyme, Jurkat cells with the inhibition of Man2c1 gene expression (AS cell) formed larger aggregates in culture, indicating an enhancement of adhesion between the cells. mRNA differential display analysis discovered up-regulation of several adhesion molecule genes in the AS cell. Because of the pivotal role played by CD54-LFA-1 interaction in immune cell interaction, this study focused on the contribution of enhanced expression of CD54 and LFA-1 to the enhanced adhesion of AS Jurkat cells. These facts, including increased binding of AS cells to ICAM-1-Fc, Mg(2+) activation of the binding of AS cells to ICAM-1-Fc and enhanced aggregation of AS cells, together with the inhibiting effect of a blocking CD11a mAb on the binding to ICAM-1-Fc and aggregation of the cells demonstrate an important contribution of enhanced CD54-LFA-1 interaction to increased adhesion between AS cells. The enhanced CD54-LFA-1 interaction also resulted in increased adhesion between AS Jurkat T cells and Raji B cells. In addition, AS cells showed cytoskeletal rearrangement. The data imply a biological significance of MAN2C1 in T-cell functioning.