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Effects of Cycloheximide and Dexamethasone on Fas - Mediated Apopthsis in Primary Human Astrocytes.

Korean Journal of Immunology.1999;21(4):361-368.

Astrocytes are major glial cells in central nervous system (CNS) and are known to express death receptors or ligands that can induce apoptosis of astrocytes or other brain cells. We have previously confirmed that cultured human astrocytes express fas and fas ligand and their expression may be regulated by various cytokines found in CNS. Because fas can rnediate cell death known as apoptosis, we investigated fas-mediated cell death in cultured human astrocytes and evaluated factors that may influence the fas-mediated apoptosis in astrocytes. Pretreatment of interferon-r and TNF-a increased cell death in astrocytes. Cell death induced by fas ligation was confirmed as apoptosis by phosphatidylserine translocation in cell membrane. Cycloheximide, protein synthesis inhibitor, potentiated fas-mediated cell death. However, buthionine sulfoxine did not potentiate fas-mediated apoptosis. Dexamethasone blocked cell death in dose-dependent and time-dependent manners. These findings collectively show that fas expressed on cultured human fetal astrocytes can induce apoptotic cell death after pretreatment of interferon-r and/or TNF-a. Therefore, the fas-fas ligand system in CNS may regulate the glial degeneration and may participate the neuronal loss in certain conditions. Furthermore, fas-mediated apoptosis of astrocytes can be potentiated by protein synthesis inhibitors and can be blocked by dexamethasone.
Apoptosis ; Astrocytes* ; Brain ; Cell Death ; Cell Membrane ; Central Nervous System ; Cycloheximide* ; Cytokines ; Dexamethasone* ; Fas Ligand Protein ; Humans* ; Ligands ; Ligation ; Neuroglia ; Neurons ; Protein Synthesis Inhibitors ; Receptors, Death Domain ; Signal Transduction

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The Effect of Ultraviolet lrradiation on Susceptibility of Listeria monocytogenes lnfection and the Production of Cytokines.

Wook LEW ; Soo Kyoung CHANG ; Kir Young KIM ; Bong Ki LEE

Korean Journal of Immunology.1999;21(4):353-360.

Although the effect of ultraviolet (UV) irradiation on immune response was reported to supress cellular immune response, the exact mechanism was not elucidated. As recent development in cytokine research progress, it is well-known that immune response is regulated by cytokines and especially cellular immune response is induced by interferon (IFN)-r and interleukin (IL)-12 which is mainly produced from lymphocytes and macrophages respectively. Therefore our purpose was to elucidate the UV effect on cellular immune response and its mechanism. We have investigated the changes of host resistance by injection of Listeria monocytogenes which is an intracellular parasite after UVB irradiation in C57BL/6 mice which is known to have relatively strong cellular immune response. In addition we also have investigated the changes in the production of IFN-r from lymphocytes and the production of tumor necrosis factor (TNF)-a and IL-12 from macrophages in mice by UVB irradiation. The increase of mouse spleen index and susceptibility of iisteria monocytogenes infection was correlated with the decreased production of IFN-r, TNF-a and IL-12, which was known to induce the suppression of cellular imrnune response.
Animals ; Cytokines* ; Immunity, Cellular ; Interferons ; Interleukin-12 ; Interleukins ; Listeria monocytogenes* ; Listeria* ; Lymphocytes ; Macrophages ; Mice ; Parasites ; Spleen ; Tumor Necrosis Factor-alpha

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IFN -r Enhances Induction of Chemokines Mig and IP10 mRNA from THP - 1 Cells Stimulated with Lipoarabinomannan.

Hwang Ho LEE

Korean Journal of Immunology.1999;21(4):343-351.

Lipoarabinomannans (LAM) is believed as a potential virulence factor of Mycobacterium tuberculosis. LAM exhibits marked differences in biological activities depending on the types, arabinofuranosyl-terminated LAM (AraLAM) derived from a rapidly growing Mycobacterium sp. and heavily mannosylated LAM (ManLAM) derived from the Erdman strain. Collaboration between macrophages and T cells, especially macrophage activation by gamma interferon (IFN-r) and chemoattraction of T cells at the very inflammatory foci would be essential in defence against M. tubercu/osis. Chemokines Mig and IP-10 are inducible by IFN-r from macrophages and have been shown to act in vitro as T cell chemoattractants. However, little is known of LAMs capacity to induce chemokines Mig and IP-10 in macrophages. In this experiment, Mig and IP10 mRNA was expressed in the delayed-type hypersensitivity (DTH) against BCG in BCG-immune mice. In some experiments, both Mig and IP-10 mRNA was evidently induced with different time courses in THP-1 cells stimulated with whole live M. tubercu/osis H37Rv (Erdman). To investigate whether Mig and IP-10 genes are differentially induced depending on the type of LAM, PCR amplification was used to detect mRNA of Mig and IP-10 from the THP-1 human monocytic cells stimulated with LAM. AraLAM, but not ManLAM, induced weakly Mig and IP-10 mRNA in the THP-1 cells. The induction of Mig and IP-10 was dependent upon the dose of AraLAM and exhibited different time courses. The mRNA for Mig and IP-10 was induced within 2 hr and 4 hr from the initiation of treatrnent and has disappeared by 8 hr and 24 hr under the experimental conditions used in this study, respectively. IFN-y at 100 U/ml, but not at 10 U/ml, was itself a good stimulus of both Mig and IP- 10 expression, and synergized with either AraLAM or ManLAM for induction of both Mig and IP-10. The expression patterns of MCP-3 were somewhat similar to those of Mig and IP10 in all of the experiments. These data indicate that IFN-r may contribute to effective macrophage function if macrophages are not fully affected by ManLAM, and chemokines Mig and IP-10 may a role in recruitment of T cells at inflammatory foci of tuberculosis.
Animals ; Chemokines* ; Chemotactic Factors ; Cooperative Behavior ; Humans ; Hypersensitivity ; Interferons ; Macrophage Activation ; Macrophages ; Mice ; Mycobacterium ; Mycobacterium bovis ; Mycobacterium tuberculosis ; Polymerase Chain Reaction ; RNA, Messenger* ; T-Lymphocytes ; Tuberculosis ; Virulence

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Induction and Expression of Chemokines and Their Receptors in Human Mast Cell Line ( HMC - 1 ).

Youn Mun HA

Korean Journal of Immunology.1999;21(4):335-342.

The mast cell is an essential effector cell in allergic inflammation through its capacity to respond to IgE dependent activation. Mast cells also participate in the modulation of physiologic processes, but the role of mast cell in these processes is still unclear. Recently, the number of structurally defined chernoattractants for leukocytes has greatly increased, owing to largely to the identification of the chemokine superfamily. In this study we examined the pattern of expression of chemokines and their receptors in HMC-1 after treatment with PMA/A23187 and/or LPS using RT-PCR and ELISA. Messenger RNA of IL-8, the representative CXC chemokine, was induced after PMA/ A23187 treatment. All of the CC chemokines tested, except eotaxin, were induced after PMA/A23187 treatment. CCR1, CXCR2, CXCR3 and CXCR4 were expressed in all test groups regardless of activation. CCR3 was expressed only at 3 hours of activation. CCR2, CCR5 and CXCR1 were not expressed in mast cell line. Production of most of chemokine proteins was not detected in resting state and increased significantly after 3 hours of activation with PMA/A23187. The effect of LPS treatment was negligible. MCP-1 protein was always produced without activation and accurnulated in a time-dependent rnanner. These data suggest that the expression of mRNA and protein of chemokines and chemokine receptors are regulated transcriptionally and translationally. Human mast cell may respond to various stimuli by producing chemokines and their receptors to regulate their function and may act autonomously or through other inflammatory cell that they recruited.
Calcimycin ; Chemokines* ; Chemokines, CC ; Enzyme-Linked Immunosorbent Assay ; Humans* ; Immunoglobulin E ; Inflammation ; Interleukin-8 ; Leukocytes ; Mast Cells* ; Receptors, Chemokine ; RNA, Messenger

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Identification of Inducible Murine Mast Cell Genes by Suppression PCR - Based Subtractive Hybridization.

Youn Mun HA

Korean Journal of Immunology.1999;21(4):327-334.

The mast cell is an essential effector cell in allergic inflammation through its capacity to respond to IgE dependent activation. Mast cells also participate in the modulation of physiologic processes, but the role of mast cell in these processes is still unclear. Recently, the number of structurally defined chernoattractants for leukocytes has greatly increased, owing to largely to the identification of the chemokine superfamily. In this study we examined the pattern of expression of chemokines and their receptors in HMC-1 after treatment with PMA/A23187 and/or LPS using RT-PCR and ELISA. Messenger RNA of IL-8, the representative CXC chemokine, was induced after PMA/ A23187 treatment. All of the CC chemokines tested, except eotaxin, were induced after PMA/A23187 treatment. CCR1, CXCR2, CXCR3 and CXCR4 were expressed in all test groups regardless of activation. CCR3 was expressed only at 3 hours of activation. CCR2, CCR5 and CXCR1 were not expressed in mast cell line. Production of most of chemokine proteins was not detected in resting state and increased significantly after 3 hours of activation with PMA/A23187. The effect of LPS treatment was negligible. MCP-1 protein was always produced without activation and accurnulated in a time-dependent rnanner. These data suggest that the expression of mRNA and protein of chemokines and chemokine receptors are regulated transcriptionally and translationally. Human mast cell may respond to various stimuli by producing chemokines and their receptors to regulate their function and may act autonomously or through other inflammatory cell that they recruited.
Calcimycin ; Chemokines ; Chemokines, CC ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin E ; Inflammation ; Interleukin-8 ; Leukocytes ; Mast Cells* ; Polymerase Chain Reaction* ; Receptors, Chemokine ; RNA, Messenger

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Oral Tolerance in Active Fatal Anaphylaxis.

Hern Ku LEE

Korean Journal of Immunology.1999;21(4):319-325.

We have investigated whether oral administration of ovalbumin (OVA) could prevent active systemic anaphylaxis to the antigen. Oral tolerance was induced by a single feecfing with 40 mg OVA before, but not after, sensitization characterized by diminished OVA-specific IgE and IgG responses. Feeding 15 mg OVA suppressed anaphylaxis and antibody responses to a lesser extent. Spleen cells from tolerant donors were incapable of transferring the tolerance to naive recipients. Pretreatment of cyclophosphamide (100 mg/kg) 2 days before OVA feeding did not restore the tolerance. Furthermore, in vitro cell mixing studies showed that the proliferation of spleen cells from OVA- sensitized donors was not inhibited by the addition of spleen cells from tolerant donors, arguing against the role of suppressor cells. Anergy was demonstrated by the ability to reverse the tolerant state after culturing tolerant cells in the presence of IL-2. These findings indicate that only a high-dose (40 mg) feeding OVA was found to be effective in inducing tolerance in this experimental system, and demonstrate anergy as the mechanism underlying oral tolerance to systemic anaphylaxis.
Administration, Oral ; Anaphylaxis* ; Antibody Formation ; Cyclophosphamide ; Humans ; Immunoglobulin E ; Immunoglobulin G ; Interleukin-2 ; Ovalbumin ; Ovum ; Spleen ; Tissue Donors

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Control of Scarring in Adult Wounds using Antisense Connective Tissue Growth Factor Ollgodeoxynucleotides.

Hun Taeg CHUNG

Korean Journal of Immunology.1999;21(4):311-317.

Adult wounds heal with scar formation, whereas fetal wounds heal without scarring and with a lesser inflammatory and cytokine response. Recently connective tissue growth factor (CTGF) is known to play an important role in wound healing. We reasoned that a strategy employing antisense oligodeoxynucleotides (ODN) complementary to CTGF mRNA by topical application of the ODN on the skin wound. Phosphorothioation of ODN to retard their degradation. When antisense CTGF ODN were applied on the wound site, there was a marked reduction of scarring compared with a control wound site. This effect of antisense CTGF ODN on scar forrnation was associated with decreased expression of the CTGF gene. However, sense CTGF ODN had no effect on the expression of the CTGF gene. In addition, control wounds healed with excessive fibrosis compared with the antisense-treated wounds. In conclusion, our results indicate that antisense CTGF ODN could be used for ameliorating scar formation during wound healing.
Adult* ; Cicatrix* ; Connective Tissue Growth Factor* ; Connective Tissue* ; Fibrosis ; Humans ; Oligodeoxyribonucleotides ; RNA, Messenger ; Skin ; Wound Healing ; Wounds and Injuries*

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Production of CD44v6 Antibody Fragments and Comparision of Their Speciticities.

Insook HAN ; Seok Kil ZEON ; Kwan Kyu PARK

Korean Journal of Immunology.1999;21(4):303-309.

CD44v6 was known as tumor marker for tumor progression and metastasis in various kinds of carcinomas. The CD44v6 monoclonal antibody was produced by cell cultures or mouse ascite fluids using CD44v6 hybridoma cells, and its immunogloburin G (IgG) was purified by Protein A column. Using immobilized ficin and cysteine, the antibody fragment Fab was produced and purified by Protein A. Four CD44v6 scFv molecules were produced from the recombinant DNA and phage antibody technology and prurified by His-tag affinity chromatography. In order to inspect the function and specificity of each antibody molecule, western-blotting and ELISA against CD44v5-6 recombinant proteins and irnmunodetection in human ovarian carcinomas were estabilished. The results showed that immunodiagnosis did not distinguish the types of antibody fragments, but western-blotting and ELISA results did show some difference of their specificities and biological properties. These studies will contribute as a model study for the immunodiagnosis and therapy using the IgG, Fab and scFv of CD44v6 antibody to obtain the early detection of tumor progression and metastasis using immunoscintigraphy.
Animals ; Bacteriophages ; Cell Culture Techniques ; Chromatography, Affinity ; Cysteine ; DNA, Recombinant ; Enzyme-Linked Immunosorbent Assay ; Ficain ; Humans ; Hybridomas ; Immunoglobulin Fragments* ; Immunoglobulin G ; Immunologic Tests ; Mice ; Neoplasm Metastasis ; Recombinant Proteins ; Sensitivity and Specificity ; Staphylococcal Protein A

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Production of IL-15 and lts Functional Study in Mouse Splenocyte Activation.

In Sook HAN ; Jong Wook PARK

Korean Journal of Immunology.1999;21(4):297-302.

After the synthesis of IL-15 cDNA from the total RNA of mouse spleen, it was inserted into the prokaryotic expression vector, pRseta, and eukaryotic expression vector, pcDNA3.0, respectively. Subsequently, the insertion of gene and open reading frame were confirmed by sequencing of each plasmid, respectively. Using pRseta- IL-15 plasmid, the recombinant IL-15 protein was induced by IPTG under BL21 (DE 3) host cells and recombinant IL-15 was expressed at 14.5 KDa with time. Then, IL- 15 was separated by His-tag affinity chromatography and analyzed by SDS-PAGE to yield soluble IL-15 at 14.5 KDa as monomer and 29.0 KDa as dimer. In order to inspect the function and contribution of IL-15, the in vitro experiment was established using mononuclear cells separated from the mouse spleen. After 48h exposure of PHA to mouse splenocyte and 24h treatment with recombinant IL-15, the effects of cytokine inductions inspected against IL-2, IL-6, IL-10, IL-12, IFN-r, and GM-CSF. The results showed that comparing with the control, IL-6 increased, IL-2, IL-12 and IFN-r increased and similar, and GM-CSF decreased. In addition, the direct injection of pcDNA3.0-IL-15 plasmid into mice gave the similar results to in vitro studies. Namely, IL-6 and IL-12 increased, and IL-2, IFN-r and GM-CSF were similar or decreased. IL-10 was not induced in in vitro and in vivo experiments. These results suggested that the IL-15 induce the splenocyte activation and can be an important factor in proliferation and fuction recovery of weakened T-cell.
Animals ; Chromatography, Affinity ; DNA, Complementary ; Electrophoresis, Polyacrylamide Gel ; Granulocyte-Macrophage Colony-Stimulating Factor ; Interleukin-10 ; Interleukin-12 ; Interleukin-15* ; Interleukin-2 ; Interleukin-6 ; Isopropyl Thiogalactoside ; Mice* ; Open Reading Frames ; Plasmids ; RNA ; Spleen ; T-Lymphocytes

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Chain Dominance Study of Monoclonal MRL - lpr / lpr Anti - DNA Autoantibodies by Ultraviolet Cross - linking Experiment.

Hyung Il KIM ; Young Ju JANG ; Soo Youn BAEK ; Sun PARK ; Jae Seung KANG ; Sang Moo LIM ; Young Tai KIM

Korean Journal of Immunology.1997;19(1):7-16.

No abstract available.
Autoantibodies* ; DNA*

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