Based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (Bacmid) which propagated in Escherichia coli, the Bac-to-Bac System provides a rapid and efficient method to generate recombinant baculoviruses and is widely used for high level expression of heterologous proteins. And the efficiency of recombinant baculovirus infecting cells plays an important role on the protein expression. In this study, we introduced an EGFP expression cassette driven by polyhedrin promoter into the p74 locus of Bacmid by homologous recombination. The target Bacmid-egfp was then transformed into E. coli DH10B containing the transposition helper plasmid to gain a new transposition receipt strain E. coli DH10Bac-egfp. Because of the intact attTn7 sites and lacZ', target gene cloned in a pFastBac vector can be transposed into the Bacmid-egfp shutter vector to construct recombinant baculovirus, which would allow the tracing of the target protein expression and the recombinant Bacmid transfection or recombinant baculoviral infection under fluorescence microscopes. Recombinant virus Bac-egfp-DsRed was constructed by transposing DsRed into the Bacmid-egfp in E. coliDHl0Bac-egfp, and the Sf9 cells infected with the recombinant virus expressed DsRed and EGFP efficiently. Another protein IL-6 fused with 6 x his tag was expressed and purified sucessfully from Sf9 cells infected with recombinant virus Bac-egfp-6 x his-IL6 constructed by the improved Bac-to-Bac system.
Animals ; Baculoviridae ; genetics ; Green Fluorescent Proteins ; genetics ; Interleukin-6 ; biosynthesis ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Spodoptera

To investigate the influence of the thalidomide on the growth of multiple myeloma cells from untreated, relapsed or refractory patients and summarize its mechanisms, thalidomide influence on colony growth of untreated, relapsed or refractory multiple myeloma cells cultured by semisolid methylcellulose was observed. The level of interleukin-6 (IL-6) autosecreted by myeloma cells was tested by IL-6-dependent cell line when myeloma cells were treated with thalidomide at 200 microgram/ml, and in the same concentration of thalidomide the expression of IL-6 receptor were tested by flow cytometry. Results showed that colony growths of myeloma cell from untreated and relapsed or refractory patients were all colonies were inhibited when treated by thalidomide up to 75 microgram/ml or 100 microgram/ml concentration. The inhibition was concentration-dependent, higher concentration cause more inhibition. After treatment with thalidomide at 200 microgram/ml, the concentrations of IL-6 secreted by myeloma cells were (148.5 +/- 96.7) microgram/ml, and the levels of IL-6 receptor expressed on the cell surface were 16.7% and 20.2% in untreated and relapsed or refractory patients, respectively, and those were significantly lower than those levels in the cells before exposure to thalidomide. It was concluded that thalidomide can inhibit growth of both relapsed or refractory cells and untreated myeloma cells in vitro. Therefore, it can be used to treat untreated multiple myeloma patients. Inhibiting tumor cells secreting level of IL-6 and reducing the expression of IL-6 receptor on myeloma cell surface is one of the mechanisms for thalidomide to remedy multiple myeloma patients
Angiogenesis Inhibitors ; pharmacology ; Cell Division ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Interleukin-6 ; metabolism ; Multiple Myeloma ; metabolism ; pathology ; Receptors, Interleukin-6 ; biosynthesis ; Thalidomide ; pharmacology ; Tumor Cells, Cultured

Adolescent ; Adult ; Cells, Cultured ; Dendritic Cells ; immunology ; Female ; Heparin ; pharmacology ; Hepatitis B, Chronic ; immunology ; Humans ; Interleukin-12 ; biosynthesis ; Interleukin-6 ; biosynthesis ; Male ; Middle Aged ; Phenotype

OBJECTIVETo study the impact of an agonist anti-CD(40) monoclonal antibody 5C11 on the induction and biological characteristics of leukemic dendritic cells.

METHODSCombinations of 5C11 and different cytokines were used to induce differentiation of leukemic blasts into dendritic cells. Morphology was observed by light microscopy. Surface antigens of the induced cells were analyzed by fluorescence-activated cell sorting (FACS), the yields of dendritic cell by cell counting, the levels of IL-6 and IL-12 by ELISA, T cell proliferating activity by allo-mixed lymphocyte reaction (MLR) in vitro. Allogeneic T cells were stimulated with leukemic dendritic cells and T-cell cytotoxicity was measured by MTT assay.

RESULTSWhen cultured with combinations of 5C11 and different cytokines, the leukemic cells isolated from the patients could differentiate into dendritic cells. The morphology showed typical features of dendritic cells, which expressed high levels of CD(40), CD(80) and CD(86). In comparison with the original leukemia cells, the leukemic dendritic cells secreted less IL-6 but more IL-12 (P < 0.05). The leukemic dendritic cells were potent to stimulate the proliferation of allogeneic T cells, and the latter was able to lyse the original leukemia cells.

CONCLUSIONLeukemic blasts could be induced to differentiate into functional dendritic cells. It may be of great value in the adoptive immunologic therapy of leukemia.

Antibodies, Monoclonal ; immunology ; CD40 Antigens ; physiology ; Cell Differentiation ; Dendritic Cells ; immunology ; Humans ; Immunophenotyping ; Immunotherapy ; Interleukin-12 ; biosynthesis ; Interleukin-6 ; biosynthesis ; Leukemia ; immunology ; pathology ; therapy

This article reviews the structure, physical and chemical characteristics of bone morphogenetic protein, summarized the effects of bone morphogenetic protein on hematopoiesis in embryo and adult animal, discussed the possible mechanisms and pointed out the theoretic and practical significance on this research work.
Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Bone Morphogenetic Proteins ; pharmacology ; Hematopoiesis ; drug effects ; Humans ; Interleukin-1 ; biosynthesis ; Interleukin-6 ; biosynthesis ; Research ; trends

OBJECTIVEThis study aimed at investigating effects of exogenous interleukine-10 (IL-10) on IL-6 and intercellular adhesion molecular (ICAM-1) expression in inflamed gingival tissue.

METHODSThe expression of IL-6 and ICAM-1 was examined using immunohistochemical techniques. Inflammatory gingival tissue treated with IL-10 was taken as the experimental group and the same patient's inflammatory gingival tissue without treatment of IL-10 was included into the control group.

RESULTSIL-6 expression was found mainly in monocytes, macrophages, lymphocytes, endotheliocytes and fibroblasts. The expression of ICAM-1 was found mainly in epithelial cells, monocot-macrophages, lymphocytes, endotheliocytes and fibroblasts. The immunohistochemical optical density (IOD) of the expression of IL-6 and ICAM-1 was detected by using Image-Proplus software, and the results showed that the expression in the experimental group differed significantly from that in the control group.

CONCLUSIONThe exogenous IL-10 may down-regulate IL-6 and ICAM-1 expression in inflammatory gingival tissue.

Adult ; Down-Regulation ; Female ; Gingiva ; metabolism ; Gingivitis ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; biosynthesis ; genetics ; Interleukin-10 ; pharmacology ; Interleukin-6 ; biosynthesis ; genetics ; Male ; Middle Aged

OBJECTIVETo investigate the expression and the localization of Cathepsin K and IL-6 mRNA in root-resorbing tissue and to elucidate the molecular changes and mechanism of root resorption induced by tooth movement.

METHODSRats were subject to experimental tooth movement to induce root resorption. In situ hybridization was performed to identify the cells in root-resorbing tissue that produced Cathepsin K or IL-6 the difference of CK mRNA or IL-6 mRNA expression between root resorption group and control group was calculated by t-test.

RESULTSCathepsin K mRNA was highly and selectively expressed in multinuclear odontoclast and IL-6 mRNA expressed in fibroblast, osteoblast, osteocyte and cementoblast. The expression of Cathepsin K mRNA and IL-6 mRNA in root-resorbing tissue increased evidently compared with the normal periodontium.

CONCLUSIONSOdontoclast in the root-resorbing tissue expresses Cathepsin K mRNA that participates in proteolysis during root resorption. IL-6 plays a very important role in the root resorption as a multifunctional cytokine.

Animals ; Cathepsin K ; Cathepsins ; biosynthesis ; genetics ; Interleukin-6 ; biosynthesis ; genetics ; Male ; Osteoclasts ; enzymology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Tooth Movement Techniques ; Tooth Resorption ; enzymology

OBJECTIVETo investigate the neuro-immune regulatory mechanism of Heart Benefiting recipe (HBR), an effective recipe for treatment of Alzheimer's disease (AD).

METHODSUsing immunohistochemical and RT-PCR methods, the neuro-immunological pathological changes in the AD rat model induced by beta-amyloid protein (A beta1-40) via lateral cerebral ventricle injection, including mainly the glial fibrillary acidic protein expression and inflammatory cytokines IL-1beta, IL-6mRNA and beta-amyloid protein precursor (APPmRNA) gene expression were studied. And the effects of HBR on these parameters were observed.

RESULTSDeposition of A beta in cerebral tissue could induce activation of stellate glial cells and abnormal increased levels of inflammatory cytokines (IL-1beta and IL-6mRNA), also the elevation of APPmRNA level. HBR could effectively control the above-mentioned pathological changes.

CONCLUSIONHBR could effectively control the inflammation and the A beta immune cascade reaction in brain of AD patients, it is one of the important therapeutic mechanisms of the recipe.

Alzheimer Disease ; chemically induced ; metabolism ; Amyloid beta-Peptides ; Amyloid beta-Protein Precursor ; biosynthesis ; genetics ; Animals ; Brain ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Interleukin-1 ; biosynthesis ; genetics ; Interleukin-6 ; biosynthesis ; genetics ; Male ; Neuroprotective Agents ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats

OBJECTIVETo investigate the role of IL-17 in the overproduction of autoantibodies and IL-6 overexpression by peripheral blood mononuclear cells (PBMC) of lupus nephritis (LN) patients.

METHODSFifteen consecutively hospitalized LN patients were selected as subjects and 15 healthy adults as normal controls. PBMC were obtained by Ficoll density gradient centrifugation. IgG, anti-dsDNA antibody and IL-6 protein levels were assessed using enzyme-linked immunosorbent assays (ELISA) on the supernatant of cultured PBMC of LN patients or normal controls. IL-6 mRNA levels in PBMC were measured using reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSIn medium culture, IgG, anti-dsDNA and IL-6 protein levels of the supernatant of PBMC from LN patients were significantly higher than those from normal controls (1492.1 +/- 73.2 ng/ml vs 636.7 +/- 51.9 ng/ml for IgG, 306.6 +/- 53.7 IU/ml vs 95.8 +/- 11.6 IU/ml for anti-dsDNA and 50.92 +/- 15.92 ng/ml vs 1.77 +/- 0.73 ng/ml for IL-6, all P < 0.001). In LN patients, IgG, anti-dsDNA and IL-6 protein levels were higher in the supernatants of PBMC in the IL-17-stimulated culture than the medium culture, but in normal controls, only the IL-6 protein levels were significantly higher. The increase in IgG, anti-dsDNA and IL-6 protein levels induced by IL-17 was dose-dependent and could be completely blocked by IL-17 monoclonal antibody mIgG(28) and partially blocked by dexamethasone. Similarly, IL-6 mRNA overexpression of PBMC in LN patients or normal controls induced by IL-17 was both dose- and time-dependent. During medium culture, IL-6 mRNA levels in LN patients were significantly higher than those in normal controls (1.80 +/- 0.11 vs 0.36 +/- 0.07). During stimulation with IL-17, IL-6 mRNA levels in LN patients were higher than those in normal controls (3.21 +/- 0.24 vs 1.30 +/- 0.14, P < 0.05) and also significantly higher when comparing the stimulated culture with the medium culture either in LN patients or normal control.

CONCLUSIONSIL-17 may play an important role in the pathogenesis of LN through the induction of IgG, anti-dsDNA overproduction and IL-6 overexpression of PBMC in LN patients.

Adolescent ; Adult ; Antibodies, Antinuclear ; biosynthesis ; Autoantibodies ; biosynthesis ; Female ; Humans ; Immunoglobulin G ; biosynthesis ; Interleukin-17 ; pharmacology ; Interleukin-6 ; biosynthesis ; genetics ; Leukocytes, Mononuclear ; metabolism ; Lupus Nephritis ; immunology ; Male ; RNA, Messenger ; analysis

OBJECTIVESevere pneumonia is one of the common severe diseases in children. Increasing evidences show that immune response greatly contribute to severe pneumonia. Dendritic cells (DC) are the important antigen presenting cells in the lung. To study the role of dendritic cells in development of severe pneumonia in children, the authors measured the number of mature DC in bronchoalveolar lavage fluid (BALF), and evaluated the relationship among IL-12, pro-inflammatory cytokines and clinical scores.

METHODSThe following 3 groups of children were enrolled in this study: severe pneumonia group: 27 children with severe pneumonia treated between November 2002 and May 2003 in PICU; mild pneumonia group: 30 children with mild pneumonia in department of pulmonology; control group: 29 children without pneumonia but receiving ventilator treatment for chest surgery. Mature DC in BALF was determined in severe pneumonia group and the control group on the day of tracheal intubation for mechanical ventilation. Acute lung injury scores and severe disease scores were evaluated in children with severe pneumonia and mild pneumonia. All children's serum levels of TNF-alpha, IL-6 and IL-12 were measured by using ELISA within 24 hours after admission. SPSS version 11.5 was used for statistical analysis.

RESULTS(1) The percent of mature DC in children with severe pneumonia was significantly higher when compared with the control group on the first day after ventilation [14.2 (3.9 - 51.8)] vs. [1.3 (0.2 - 22.5)] (Z = 5.44, P < 0.01). (2) In severe pneumonia group, the concentration of serum IL-12 [117.0 (79.9 - 159.4) ng/L], TNF-alpha [90.6 (52.2 - 185.9) ng/L], IL-6 [128.7 (73.3 - 793.8) ng/L] were significantly higher than those in mild pneumonia group where the values were [71.6 (19.4 - 196.8)], [26.6 (2.5 - 113.9)], and [39.9 (7.8 - 82.5)] (P < 0.01), and the control group [6.4 (12.2 - 92.0)], [6.4 (1.8 - 91.9)], and [23.0 (6.4 - 54.2)] (P < 0.01). Serum IL-12, TNF-alpha and IL-6 levels in children with mild pneumonia were higher than those of control group (P < 0.01). (3) The percent of mature DC was increased with the serum level of IL-12 (r = 0.48, P < 0.01), TNF-alpha (r = 0.58, P < 0.01), IL-6 (r = 0.51, P < 0.01) and lung injury scores (r = 0.39, P < 0.05), but it did not correlate with severe disease scores (r = -0.11, P > 0.05).

CONCLUSIONSThere is excessive production of pro-inflammatory cytokines and over-stimulation of lung dendritic cells in children with severe pneumonia. Over-stimulation of lung dendritic cells, the increased serum levels of IL-12, TNF-alpha, IL-6 and the severity of pneumonia may suggest that DC plays an important role in pathogenesis of severe pneumonia in children.

Bronchoalveolar Lavage Fluid ; cytology ; Child, Preschool ; Cytokines ; biosynthesis ; blood ; Dendritic Cells ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Infant ; Interleukin-12 ; biosynthesis ; Interleukin-6 ; biosynthesis ; Male ; Pneumonia ; immunology ; pathology ; physiopathology ; Severity of Illness Index ; Tumor Necrosis Factor-alpha ; biosynthesis