In order to study the functions of migration inhibitory factor (MIF) as macrophage activating cytokine and to investigate the possibility of MIF cDNA as gene therapeutic agent or adjuvant, we produced recombinant MIF (rMIF), anti-MIF antibody and pcDNA I plasmid containing mMIF cDNA (mMIF plasmid). We have investigated the effects of recombinant mMIF or mMIF plasmid on the expression of immune response-related gene in the mouse peritoneal macrophage or splenocyte. Recombinant mMIF produced by Baculovirus expression system was biologically active; it increased mRNA expression of tumor necrosis factor (TNF)-a, Interleukin (IL)-1, IL-6, granulocyte monocyte-colony stimulating factor (GM-CSF), nitric oxide synthase (NOS), Fas and Bcl-x when applied to the cultures of mouse peritoneal macrophage. Anti-mMIF antibody blocked these effects of mMIF on macrophage. Plasmid DNA carrying MIF cDNA inoculated into mouse peritoneal cavity also increased mRNA transcriptions from TNF, IL-1, IL-6, IL-12, GM-CSF, NOS genes of peritoneal macrophage. It enhanced proliferation of splenocyte stimulated with phorbol myristate acetate and IL-2 mRNA expression of splenocytes. Frorn these results, we conclude that rMIF is a strong macrophage activating factor and especially MIF plasmid can be used as an immune potentiating DNA drug in gene therapy for cancer or DNA adjuvant in vaccination in future.
Animals ; Baculoviridae ; DNA ; DNA, Complementary* ; Genetic Therapy ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granulocytes ; Interleukin-1 ; Interleukin-12 ; Interleukin-2 ; Interleukin-6 ; Interleukins ; Macrophages* ; Macrophages, Peritoneal ; Mice ; Nitric Oxide Synthase ; Peritoneal Cavity ; Plasmids ; RNA, Messenger* ; Tetradecanoylphorbol Acetate ; Tumor Necrosis Factor-alpha ; Vaccination

PURPOSE: CD5+ B (B1) cell is a subpopulation of B cells and CD5+ B cells constitute a large fraction of B cells in neonates. CD5 B cells are closely related with autoimmune diseases but the roles and functions in neonates are still unknown. The quantitative changes of CD5+ B cells in neonatal infections were examined to investigate the involvement of CDS+ B cells in neonatal immune reaction to general immunologic stimuli such as infections. Methods: Ten normal neonates and eight neonates with acute febrile diseases were studied. Venous blood was drawn and mononuclear cells were separated by Ficoll-Hypaque. Half was double-stained with FITC-conjugated anti-CD5 and PE-conjugated anti-CD19, and another half with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8. Stained samples were analyzed using fluorescent-activated cell sortor. ...continue...
Autoimmune Diseases ; B-Lymphocytes* ; Humans ; Infant, Newborn

14-3-3 proteins are cytoplasmic proteins of about 29 kDa and have a minimum of seven isoforms. This protein is important in signal transduction with the ability of binding with phosphoserine of many signalling proteins. We expressed 14-3-3 protein tagged with 6 histidine residues in E. coli and purified the protein by nickel affinity chromatography. Using this purified protein as an antigen, we made rabbit antisera and mouse monoclonal antibodies to 14-3-3 zeta isoform. We subcloned cDNA of 14-3-3 zeta isoform derived from HeLa cell lamda gt 11 library into an E. coli expression vector which is designed to express heterologous protein with N- terminal 6 hidtidine tag. BALB/c mice were immunized with purified 14-3-3 protein and the hybridoma clones which produce monoclonal antibodies angainst 14-3-3 protein were selected. These monoclonal antibodies reacted with the recombinant protein expressed in E. coli as well as the 29-kDa native protein in various cell lines. However, they did not immunoprecipitate 14-3-3 protein. The monoclonal antibodies produced in this study can be valuable tools for the identification of the 14-3-3 in signal transduction study.
14-3-3 Proteins ; Animals ; Antibodies, Monoclonal ; Cell Line ; Chromatography, Affinity ; Clone Cells ; Cytoplasm ; DNA, Complementary ; Escherichia coli* ; Escherichia* ; HeLa Cells ; Histidine ; Humans* ; Hybridomas ; Immune Sera ; Mice ; Nickel ; Phosphoserine ; Protein Isoforms ; Signal Transduction ; Staphylococcal Protein A

Taxol, an anticancer drug, blocks cell division by stabilizing microtubules. However, taxol has distinct cell-cycle-independent effects. For example, taxol and bacterial LPS induce strikingly similar responses in murine microglial cells. Here, we report that taxol, like LPS, provides a ""second"" signal for murine microglial cell activation to induce tumoricidal activity. Tumoricidal activity determined by MTT assay appeared that taxol or LPS alone weakly activated microglial cells to kill P815 mastocytoma cells, whereas combinations of taxol or LPS with IFN-r synergized to activate macrophages to lyse tumor cells in a dose dependent manner. Secretion of nitric oxide (NO) correlated with tumor cell killing, and the activated microglial cells failed to kill tumor cell targets in the presence of N'-monomethyl-L-arginine (N'MMA), a competitive inhibitor of NO synthase (NOS). Treatment of the cells with anti-TNF-a neutralizing antibodies clearly blocked taxol plus IFN-r induced tumoricidal activity as well as NO production. Collectively, the data illustrate the potential for taxol to activate microglial cell mediated-antitumor mechanisms in addition to its better characterized role as an anti-mitotic agent.
Antibodies, Neutralizing ; Cell Division ; Homicide ; Macrophages ; Mastocytoma ; Microglia ; Microtubules ; Nitric Oxide Synthase ; Nitric Oxide* ; Paclitaxel*

The systematic study of products from bacteria and fungi has led to the development of two immunosuppressive drugs, cyclosporin A and FK 506 (tacrolimus) which are useful to suppress adaptive immune responses to the grafted tissue. However, they affect all immune responses indiscriminately and are both toxic to kidneys and other organs. To facilitate the development of immunosuppressor to block the T cell receptor (TcR)-mediated signaling cascade specifically, a novel Jurkat T cell transfectants, JK NFAT-SEAP were generated in which the expression of the secreted alkaline phosphatase (SEAP) is driven by the multiple NFAT binding sites plus minimal IL-2 promoter. Upon stimulation with ionomycin or anti-TcR mAb OKT3 in the presence of PMA, these transfectants secreted high level of SEAP into the medium, which was conveniently analyzed by SEAP analysis. The secretion of SEAP was effectively inhibited by cyclosporin A or FK 506 at the concentration of [10 ' ug/ml], [10 ug/ml] respectively. JK NFAT-SEAP transfectants will provide two major advantages for the development of a novel immunosuppressor. First, analysis of SEAP secreted into the culture medium by SEAP analysis enables us to test a large number of samples within a short period of time. Second, Usage of IL-2 promoter for the expression of SEAP makes us identify bioproducts to target specifically on TcR-mediated signaling pathway.
Alkaline Phosphatase ; Bacteria ; Binding Sites ; Cell Line* ; Cyclosporine ; Fungi ; Interleukin-2 ; Ionomycin ; Kidney ; Mass Screening* ; Muromonab-CD3 ; Receptors, Antigen, T-Cell ; T-Lymphocytes* ; Tacrolimus ; Transplants

It has been reported that the stimulated T lymphocytes might secret a neutrophil survival factor. Thus the goal of study was to determine which molecules are the neutrophil survival factors secreted from the phytohaemagglutinin (PHA)-stimulated T lymphocytes. Human peripheral blood T lymphocytes and neutrophils were isolated by Ficoll-paque density sedimentation from heparinized blood of healthy adult donors. The purity of T lymphocytes and neutrophils were more than 90% and 95%, respectively. The maximal effective condition for the neutrophil viability-sustaining activity was 1 ug/ml af PHA in 12 hours incubation with T lymphocytes. The effect of PHA-stimulated T lymphocyte conditioned medium (TCM) on the neutrophils were used for the comparison with PHA-nonstimulated TCM or enriched medium alone. Neutrophil viability-sustaining activity with PHA-stimulated TCM for 24 hours incubation was significantly higher than other groups (85+/-11 vs 43+/-5 vs 916%; p<0.01). In the analysis of the primary data, the good candidates for the neutrophil viability-sustaining factor were granulocyte/monocyte colony stimulating factor (GM-CSF) and interleukin-8 (IL-8). They were used in the bioassay and antibody neutralization of cytokine activity. ...continue...
Adult ; Biological Assay ; Colony-Stimulating Factors ; Culture Media, Conditioned ; Granulocyte-Macrophage Colony-Stimulating Factor ; Heparin ; Humans ; Interleukin-8 ; Lymphocytes ; Neutrophils* ; T-Lymphocytes ; Tissue Donors

Panax ginseng (PG) has been used as an important analeptic in traditional medicine. This study was purposed to investigate the effect of PG on immune responses induced by glucocorticoid in mice. PG solution was injected into CV6 and BL23, which are the classical acupuncture points, for 7 days after injection with glucocorticoid. And then B and T cell proliferation and cytolytic activity of natural killer (NK) cells were measured. B cell proliferation by 'H-thymidine incorporation was decreased by about 25% in control group as compared with normal group. However, B cell proliferation was significantly increased 1.8-fold in CV6 group and 2.5-fold in BL23 group as compared with normal group. T cell proliferation by H- thymidine incorporation was decreased by about 15% in control group as compared with normal group. On the other hand, T cell proliferation was significantly increased 1.9-fold in CV6 group and 2.3-fold in BL23 group as cornpared with normal group. Furthermore in purified T cell, the proliferation was furtherly increased rather than in non-purified T cell. The activity of NK cell was remarkably decreased in control group as compared with normal group. However, the activities of NK cells in CV6 and BL23 groups were recovered to the above levels of normal group. On the other hand, the activity of NK cell in the blank locus group was slightly increased compared with control group. However this increasement was not reached the levels of CV6 and BL23 groups. And in the case of purified NK cell, the cytolytic activity of NK cell was respectively increased 1.6-fold in normal group, 1.4-fold in control group, 2.0-fold in blank locus group and 2.0-fold in CV6 group and 1.4-fold in BL23 group as compared to the non-purifed NK cell. These results suggest that PG aqua-acupuncture at CV6 and BL23 may proliferate B and T cells that is suppressed by glucocorticoid, and activate NK cell activity.
Acupuncture Points ; Acupuncture* ; Animals ; Cell Proliferation ; Hand ; Killer Cells, Natural ; Lymphocytes* ; Medicine, Traditional ; Mice* ; Panax* ; T-Lymphocytes ; Thymidine

PURPOSE: The CD5 molecules are pan-T cell antigens and are found on a minor subpopulation of B cells. CD5 antigens are involed in an intracellular signal transduction as well as in an intercellular signal transduction between CDS+ T cell/CD72+ B cell by CD5/CD72 interaction. CD5 antigens are known to be participated in classic immune reactions and in this study CDS mRNA expressions by lymphocytes were examined in allergic patients controls, acute febrile infectious disease controls and normal controls to elucidate the possibility of CDS involvement in allergic immune reactions. METHODS: Fifteen allergic patients, ten patients of acute febrile infectious disease patients and ten normal controls were studied. Venous blood was drawn and mononuclear cells were separated. T cells and B cells were separated using immunomagnetic beads. Total RNA was extracted and RT-PCR (reverse transcriptase - polymerase chain reaction) was done to detect CDS antigen mRNA expression. RESULTS: 1) CDS mRNA overexpressions were detected in allergic patient controls as compared to that in acute febrile infectious controls. CDS mRNA was not detected in normal controls. Semiquantitative CD5 mRNA expressions were measured as relative expressions of CD5 to GAPDH. Relative quantities of CD5 mRNA expressions were 90.656.24% in allergic patient controls and 23.76+3.58% in acute febrile infectious patients. CONCLUSIONS: CDS mRNA overexpression is a characteristic phenomenon in allergic immune reactions. From these result, CD5/CD72 pathway might be the preference immune mechanism in allergic immune reaction and the further study for the exact mechanism of CDS involvement in allergic immune reactions may be necessary
Antigens, CD5 ; B-Lymphocytes ; Communicable Diseases ; DNA-Directed RNA Polymerases ; Humans ; Hypersensitivity ; Lymphocytes* ; RNA ; RNA, Messenger* ; Signal Transduction ; T-Lymphocytes

Oxidatively modified low density lipoprotein (LDL) could contribute to one of the atherosclerotic processes through its uptake by the scavenger receptor of macrophage. The aim of the present study was to examine whether ganghaungenin has an antioxidant effect. For the approach of the aim, a lipid peroxidation of LDL, the changes of degradation and negative charge of LDL, and LDL uptake in macrophage were measured after ganghaungenin was treated in the reaction process of LDL oxidation. The effective LDL oxidation was performed with treatment of 20 pM CuSO4 for 5 hours. Quercetin, one of the well-known antioxidant, was used as a positive control. The antioxdant effect of ganghaungenin on the LDL oxidation were examined at concentrations of both 40 p,M and 80 p,M for Sh. Ganghaungenin significantly inhibited the lipid peroxidation of LDL. It inhibited the oxidation-enhanced degradation and negative charge of LDL. Even 100 p,M of it did not have any toxic effects on macrophage. It inhibited the uptake of the oxidized LDL into macrophage. These data revealed that ganghaungenin obtained from Scutellaria Baicalensis Georgi was a potent antioxidant in inhibiting the oxidative modification of LDL. Conclusively, these findings indicate that ganghaungenin in physiologic concentrations could inhibit the oxidative modification of LDL in vivo, and suggest that ganghaungenin could be also used for the atherosclerosis prevention.
Antioxidants* ; Atherosclerosis* ; Lipid Peroxidation ; Lipoproteins ; Macrophages* ; Quercetin ; Receptors, Scavenger ; Scutellaria baicalensis

The T cell antigen receptor (TcR) in combination with costimulatory signals triggered by accessory molecules present on the surface of the antigen-presenting cells (APC) regulates the activation and growth of T lymphocytes. Calyculin A and Okadaic acid is known to be an inhibitor of serine/threonine phosphatase and RK-682 specifically blocks functions of tyrosine phosphatase. To investigate roles of these inhibitors in TcR-mediated signaling cascade, chimeric molecule CD8-5 which contains the extracellular and transmembrane domains of the human CD8a molecule and the cytoplasmic tail of TcR 5 chain were stably expressed in Jurkat cell line. CD8-5 chimeric protein induced tyrosine phosphorylation of various cytoplasmic substrates and IL-2 gene expression in a NFAT dependent manner by stimulation with anti-CD8 mAb OKT8 as seen in TcR stimulation. When CD8-5 transfectants were preincubated with Okadaic acid, Calyculin or RK682, they differentially affected tyrosine phosphorylation of signaling mediators including CD8-5 molecule. When Jurkat Tag cell line was used where SV40 T antigen is stably expressed and the expression of p-galactosidase is driven by the multiple NFAT binding sites plus minimal IL-2 promoter, these phosphatase inhibitors -RK682, Calyculin A, Okadaic acid- effectively inhibited IL-2 gene expression at the concentration of 1.2832 x 10 ' M, 3.9924 x 10 M, 7.2707 x 10 M respectively. These results suggested that Okadaic acid, Calyculin or RK682 modulate TcR-proximal as well as TcR-distal signaling events during T cell activation.
Antigen-Presenting Cells ; Antigens, Viral, Tumor ; Binding Sites ; Cell Line ; Cytoplasm ; Gene Expression ; Humans ; Interleukin-2 ; Jurkat Cells ; Okadaic Acid* ; Phosphorylation ; Receptors, Antigen, T-Cell ; T-Lymphocytes ; Tyrosine