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

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

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

The aims of this study were to investigate the relationships between the production of interleukin-1 (IL-1), and IL-6 system by whole blood cells, and bone mineral density (BMD), and polymorphisms in IL-1 system and IL-6 gene in postmenopausal Korean women. The production of IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1ra), IL-6, and soluble IL-6 receptor (sIL-6r) by lipopolysaccharide-stimulated whole blood cells was measured by ELISA in 110 subjects. Serum osteocalcin, C-telopeptide of type I collagen, and BMD at lumbar spine and proximal femur were measured. IL-1alphaC(-889)T polymorphism, IL-1beta C(-511)T polymorphism, 86-base pair variable number tandem repeat polymorphism in the IL-1ra gene, and IL-6 C(-634)G polymorphism were analyzed. The production of IL-1beta correlated positively with BMD at femoral neck, whereas the production of other ILs did not correlate with BMD at the skeletal sites examined. No significant differences in the production of ILs were observed among normal, osteopenic and osteoporotic postmenopausal women, and among the different IL system polymorphisms groups studied. No correlation between bone turnover markers and the production of ILs was noted. In conclusion IL-1beta may regulate bone metabolism at femoral neck, and the IL system polymorphism do not affect the production of ILs by whole blood cells.
Aged ; Blood Cells/drug effects/immunology ; Bone Density/*genetics/*immunology ; Bone Diseases, Metabolic/blood/genetics/immunology ; Cytokines/*biosynthesis/blood ; Female ; Humans ; In Vitro ; Interleukin-1/biosynthesis/blood/genetics ; Interleukin-6/biosynthesis/blood/genetics ; Interleukins/*genetics ; Lipopolysaccharides/pharmacology ; Middle Aged ; Osteoporosis, Postmenopausal/blood/genetics/immunology ; *Polymorphism, Genetic ; Receptors, Interleukin-6/biosynthesis/blood/genetics ; Research Support, Non-U.S. Gov't ; Sialoglycoproteins/biosynthesis/blood/genetics

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 observe the role of Kupffer cells in the postburn production of TNFalpha, IL-1beta and IL-6 in severely scalded rats.

METHODS(1) The production of TNFalpha, IL-1beta and IL-6 from rat Kupffer cells stimulated by burn serum was observed. (2) The postburn change in the expression of cytokine mRNA from rat Kupffer cells was monitored. (3) The change in the plasma cytokine contents in scalded rats was determined after the application of gadolinium chloride, a specific inhibitor of Kupffer cells.

RESULTSKupffer cells could be stimulated by burn serum to release cytokines TNFalpha, IL-1beta and IL-6. The mRNA expression of TNFalpha, IL-1beta and IL-6 from rat Kupffer cells increased significantly after injury. But the postburn plasma levels of TNFalpha, IL-1beta and IL-6 decreased obviously to 34.71%, 36.99% and 33.7% of those in scalding group, respectively, after the Kupffer cell activity was inhibited.

CONCLUSIONThe plasma cytokines, i.e. TNFalpha, IL-1beta and IL-6, were primarily produced from Kupffer cells after injury in scalded rats, initiated by TNFalpha, IL-1beta and IL-6 mRNA transcription.

Animals ; Burns ; immunology ; metabolism ; Gadolinium ; pharmacology ; Interleukin-1 ; biosynthesis ; genetics ; Interleukin-6 ; biosynthesis ; genetics ; Kupffer Cells ; physiology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo observe the effect of Chinese herbs for supplementing Shen and strengthening bone (HB) on myelogenic osteoclasts formation, and gene expression of interleukin-6 (IL-6), IL-6 receptor (IL-6R) and gp130 in bone marrow.

METHODSSeventy-two healthy female SD rats of 3 months, were randomly divided into three groups, 24 in the sham-operated group (A), 24 in the ovariectomized group (B) and 24 in the after ovariectomy HB treated group (C). Bone marrow cells of 6 rats from each group were respectively collected and cultured at four time points (2nd, 4th, 6th and 12th weeks after operation). After 6 days of culture, the bone marrow cells were differentiated by Wright-Giemsa stain and TRAP stain, and total RNA in them was extracted by TRIZOL.

RESULTSBeginning from the 2nd week, the osteoclasts formation in Group B was higher than that in Group A (P < 0.05), and IL-6, IL-6R gene expression significantly increased in Group B (P < 0.05 or P < 0.01). These changes reached the peak in the 4th to 6th week, with the level maintained to the 12th week. As for comparison of Group B and C, the above-mentioned changes were significantly weakened in the latter (P < 0.05 or P < 0.01). No significant change of gp130 gene expression revealed in the whole course in either group.

CONCLUSIONHB could inhibit the myelogenic osteoclasts formation in ovariectomized rats, this effect may be correlated with, partially at least, its inhibitory effect on the over-expressed IL-6 and IL-6R gene expression in myelocytes after ovariectomy.

Animals ; Antigens, CD ; biosynthesis ; genetics ; Bone Marrow ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; Cytokine Receptor gp130 ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Granulocyte Precursor Cells ; metabolism ; Interleukin-6 ; biosynthesis ; genetics ; Isoflavones ; pharmacology ; Membrane Glycoproteins ; biosynthesis ; genetics ; Osteoblasts ; pathology ; Osteoporosis ; metabolism ; pathology ; Ovariectomy ; RNA ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Interleukin-6 ; biosynthesis ; genetics

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

To investigate the role of anti-inflammatory cytokine in acute coronary syndrome (ACS), the effect of IL-10 on expression of tissue factor (TF) induced by IL-6 in peripheral blood mononuclear cells (PBMNC) were studied. PBMNC were allowed to culture with rhIL-10 before being stimulated by rhIL-6. One-step recalcification clotting time was used to evaluate procoagulant activity (PCA) of PBMNC. The expression and activity of TF protein were determined by ELISA and cell chromogenic substrate assay. The results showed that the expression of PCA, TF protein and its activity in PBMNC increased significantly after being stimulated by rhIL-6 (P < 0.01). In PBMNC, rhIL-6-induced PCA was regulated by rhIL-10 in different doses. This effect was associated with reduction of TF protein expression and activity by rhIL-10 (P < 0.01). In conclusion, IL-10 down-regulated expression PCA and TF in PBMNC, inhibitory effect of IL-10 on expression and activity of PBMNC TF may be important protective mechanism for ACS, regulation imbalance between inflammatory and anti-inflammatory cytokines may be important factor participating in coronary thrombosis.
Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-10 ; pharmacology ; Interleukin-6 ; genetics ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; metabolism ; Recombinant Proteins ; pharmacology ; Thromboplastin ; biosynthesis

OBJECTIVE: To determine the expression of cyclooxygenase-2 (COX-2) mRNA in peripheral blood monocytes in patients with acute coronary syndrome (ACS), and to explore the effect of the expression of COX-2 mRNA in ACS. METHODS: The expressions of COX-2 mRNA in peripheral blood monocytes from 18 normal subjects and 42 ACS patients were analyzed by reverse transcription polymerase chain reaction (RT-PCR), and the monocytes from patients were incubated with celecoxib in vitro. The concentrations of interleukin-6 (IL-6) and matrix metalloproteinase-9 (MMP-9) in supernates of monocytes were measured by enzyme-linked immunosorbent assays (ELISA). RESULTS: The expression of COX-2 mRNA and the secrections of IL-6 and MMP-9 in peripheral blood monocytes in ACS patients significantly increased compared with those from normal controls [0.61 +/- 0.17 vs 0.11 +/- 0.09; (97.24 +/- 11.21) ng/L vs (22.15 +/- 6.30) ng/L; (41.20 +/- 8.41) g/L vs (11.76 +/- 4.23) g/L; all P < 0.05, respectively]. Celecoxib reduced IL-6 and MMP-9 secretion level of monocytes from ACS patients up to 48% and 50% respectively (all P < 0.05), in a concentration-dependent manner. CONCLUSION COX-2 in peripheral blood monocytes may play an important role in the acute coronary syndrome.
Aged ; Angina Pectoris ; blood ; enzymology ; Coronary Artery Disease ; blood ; enzymology ; Cyclooxygenase 2 ; biosynthesis ; genetics ; Female ; Humans ; Interleukin-6 ; biosynthesis ; Male ; Matrix Metalloproteinase 9 ; biosynthesis ; Middle Aged ; Monocytes ; enzymology ; RNA, Messenger ; biosynthesis ; genetics