Culture of human peripheral T lymphocytes with irnmobilized anti-CD3 rnAb plus IL-2 resulted in a marked proliferation and the enhancing IL-2Ra mRNA expression. The process of the T cell activation involves a series of biochemical events which ultimately lead to the proliferation and IL-2Ra mRNA expression. Although the above results have been observed, the celluar signal mechanisms between the proliferative response and the IL-2Ra mRNA expression through T cell receptor and IL-2 receptor remains unresolved. In the present study, We have used genistein (the selective PTK inhibitor) or chronic PMA treatment (depletion of intracelluar PKC activity), to investigate the role of PTK or PKC both in a synergistic proliferation and in the enhancing IL-2Ra mRNA expression by IL-2/anti-CD3. Genistein (30 ug/ml) completely blocked IL-2 induced T cell proliferation, and inhibited anti-CD3 induced T cell proliferation (93.4%). But genistein downregulated the IL-2Ra mRNA expression by IL-2, anti-CD3 and IL-2/anti-CD3. The chronic PMA treatment failed to inhibit the proliferation and the IL-2R#u mRNA expression by IL-2 alone. But PKC depleted T cells stimulated with anti-CD3 mAb showed the decrease of the proliferation (68.6%) and IL-2Ra mRNA expression. In activated with IL-2/anti- CD3, the proliferative response showed a half of reduction, but the IL-2Ra mRNA expression were not regulated. These results demonstrate that proliferative response to IL-2 appears to be dependent on PTKs activity and independent of PKC involvement, but the IL-2Ra mRNA expression may be required another signals. PTKs and PKC activity may be important in TCR/CD3 signaling. But IL-2/anti-CD3 are coupled up different signal transduction pathways responsible for the synergistic T cell proliferation and the enhancing IL-2Ru mRNA expression.
Cell Proliferation ; Genistein ; Humans ; Interleukin-2* ; Receptors, Antigen, T-Cell ; Receptors, Interleukin-2 ; RNA, Messenger* ; Signal Transduction ; T-Lymphocytes

An activation antigen, CD30 was initially identified on Hodgkin and Reed-Sternberg (H-RS) cells. CD30 expression is observed on activated, but not on resting, T and B lymphocytes. Despite of numerous studies, the functions of CD30 in physiological condition remains open question. Moreover, CD30 expression of normal B lymphocytes has been poorly documented. In this study, CD30 expression of murine B lymphocytes and its regulation was analyzed. Murine splenic B (SP-B) cells obtained by adherence were used for activation with LPS or plate-bound anti-mouse IgM. LPS stimulation resulted in B cell proliferation. However, stimulation with plate-bound anti- mouse IgM (pb anti-mlgM) induced blast cell formation but did not increase cell number. Both stimulation induced minimal expression of CD30. Substantial CD30 expression of SP-B cells was induced by IL 4, which upregulated both of proliferation and CD30 expression of activated SP-B cells. Highest level of CD30 expression was detected at day 3 of stimulation. IL 2 enhanced B cell proliferation but not CD30 expression and rather reduced IL 4-mediated upregulation of CD30 expression. These data suggest that the signaling pathway for B cell proliferation is different from that for induction of CD30 expression and IL 4 exerts a pivotal role in CD30 expression of both T and B cells. In addition, T and B cells may show distinct response to other cytokines such as IL 2 in CD30 expression.
Animals ; Antigens, CD30 ; B-Lymphocytes* ; Cell Count ; Cell Proliferation ; Cytokines ; Immunoglobulin M ; Mice ; Up-Regulation

Apoptosis has ernerged as a key mechanism for regulating the number of leukocytes at sites of inflammation. Besides withdrawal of inflammatory stimuli, apoptosis of human monocytes can be directly triggered through two cell surface molecules, Fas and the IL-4 receptor. In contrast to Fas-mediated death which utilizes reactive oxygen intermediates (ROI) as instruments of death, IL-4-induced apoptosis of monocytes was neither blocked by antioxidants, nor accompanied by elevation of cellular ROI. Moreover, PMA which upregulates protein kinase C (PKC) inhibited IL-4-, but not Fas-mediated death. These data define a ROl-dependent, PKC-resistant Fas pathway, and a ROl-independent, PKC-susceptible IL-4 pathway of apoptosis. Monocyte apoptosis triggered by depletion of inflammatory mediators resembles the IL-4 pathway. Within the context of an inflammatory site, monocyte accumulation and depletion may be susceptible to manipulation through these pathways.
Antioxidants ; Apoptosis ; Humans* ; Inflammation ; Interleukin-4* ; Leukocytes ; Monocytes* ; Oxygen ; Protein Kinase C ; Receptors, Interleukin-4

Many fungi including Penicillium, Aspergillus, Gliocladium, and Thermoascus produce an epipolythiodioxopiperazine class of fungal metabolite, gliotoxin, which contirbutes the pathogenesis of fungal infection as an immunomodulator and cytotoxic agent. This study is designed to define the mechanism by which gliotoxin exerts the cytotoxic effect of gliotoxin on human promyelocytic leukemic cells, HL-60. Gliotoxin induces the apoptosis of HL-60 cells which is characterized by the ladder pattern fragmentation of DNA. Gliotoxin induces the activation of DEVD-specific cysteine protease in a time- and dose-dependent rnanner. It also increases the phosphotransferase activities of c-Jun N-terminal kinase1 (JNK1) and p38 in gliotoxin-treated HL-60 cells. Furthermore, gliotoxin decreases the activation of transcriptional activator, actiating protein (AP-1) and NF-kB. These results suggest that gliotoxin induces the apoptotic death of HL-60 cells via activation of DEVD- specific caspase as well as mitogen activated protein kinases (MAP kinases) including JNK1 and p38, and inhibition of transcriptional activators, AP-1 and NF-kB.
Apoptosis* ; Aspergillus ; Caspase 3 ; Cysteine Proteases ; DNA ; Fungi ; Gliocladium ; Gliotoxin* ; HL-60 Cells* ; Humans ; Mitogen-Activated Protein Kinases ; NF-kappa B ; Penicillium ; Thermoascus ; Transcription Factor AP-1 ; Transcription Factors

Programrned cell death (PCD), or apoptosis, is a process by which cells die in response to specific physiological and toxicological signals. This genetically programmed form of cellular suicide is intirnately involved in many biological processes including growth, metamorphosis, embryogenesis, and oncogenesis. Cells undergoing PCD in normal and neoplasmic tissues display the following biochemical and morphological features: internucleosomal DNA fragmentation, reduced cell volume, condensed chromatin in nucleus, zeiosis and generation of apoptotic bodies containing intact organelles and plasma rnembrane. Hybridoma cell production, resulting from the fusion of myeloma cells with antibody producing spleen cells, is widely used in various fields of life science. This technique requires hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient mutant myeloma cell line as a fusion partner. When these mutants cell is treated with aminopterin plus hypoxanthine-thymidine (HAT) after the cell fusion they are selectively and efficiently eliminated remaining fused hybridoma celis. But there hasn't been any report regarding the selective elimination mechanism of this HGPRT mutant myeloma cell. By using HGPRT myeloma P3-X 63-Ag8.653 (V653) as a model system, this study demonstrated that PCD was induced by aminopterin treatment of this V653 cell line. And the sequential ultrastructural changes during this death process were observed by using electron microscope. When V653 cells were incubated with 0.4 uM aminopterin, DNA fragmentation was detectable after 3 hours and peaked between 12 and 18 hours of aminopterin treatment and the cell viability decreased in a time dependant manner. V653 cells incubated with amiopterin showed following ultrastructural changes during the death process. Dilatation of rough endoplasmic reticulum (RER) and detachment of ribosomes were the earliest ultrastructural changes and first seen after 30 minute incubation. Dilatation of perinuclear cisternae began to appear after 1 hour and deformation of nucleoplasm such as decreased electron density of perinuclear heterochromatin and increased electron density of euchromatin were seen after 3 hours. Increased electron density of cytoplasm, decreased cell volume, condensation of chromatin and apoptotic bodies were observed in many cells after 9 hours but vacuolation by severe dilatation of RER was seen in some cells. 24 hours after incubation with aminopterin, many cells showed typical form of apoptosis characterized by cell shrinkage, increased electron density of cytoplasm and apoptotic bodies. On the contrary, some cells showed different type of cell death characterized by increased cell volume, decreased electron density of cytoplasm, severely dilated RER and apoptotic bodies. In both types of cells, mitochondrial cristae and limiting membrane of mitochondria are comparatively well preserved. In other cells, nuclei did not show significant changes but there were deformations of mitochondria such as markedly increased electron density and formation of lamella bodies. The death process of V653 cell was not synchronized among cells. The results of this study proved that aminopterin-induced selective elimination of fusion partner V653 myeloma cell is due to PCD. The earliest ultrastructural changes observed in this process were dilatation of RER and detachment of ribosomes. And there were two distinct morphological types in the PCD.
Aminopterin* ; Animals ; Apoptosis* ; Biological Processes ; Biological Science Disciplines ; Carcinogenesis ; Cell Death ; Cell Fusion ; Cell Line ; Cell Size ; Cell Survival ; Chromatin ; Cytoplasm ; Dilatation ; DNA Fragmentation ; Embryonic Development ; Endoplasmic Reticulum, Rough ; Euchromatin ; Female ; Guanine ; Heterochromatin ; Hybridomas ; Hypoxanthine ; Hypoxanthine Phosphoribosyltransferase ; Membranes ; Mice* ; Mitochondria ; Organelles ; Plasma ; Pregnancy ; Ribosomes ; Spleen ; Suicide ; Transferases

To generate drug delivery vector to locales in the body, genetic engineering and expression techniques have been used to produce antibody avidin fusion proteins. Chicken avidin has been fused to mouse-human chimeric IgG3 immediately after the hinge with a flexible linker (H-Flex-Av) and at the end of CH2 (CH2-Av). Fusion heavy chains were expressed with the expected molecular weight, assembled as H2L2 forms with a co-expressed light chain, and were secreted. The expression level of H- Flex-Av was 1~10 ug/ml/10(8)/24 hrs, but that of C2-Av was a very little (0.08~0.9 ug/ ml/10(8)/24 hrs). The resulting H-Flex-Av and CH2-Av fusion proteins continued to bind antigen dansyl and also bound biotinylated bovine serum albumin; both H-Flex-Av and CH2-Av had shown to retain 3-4 times higher relative affinity than that of CH3-Av in ELISA. Importantly the fact that both avidin fusion proteins had a higher relative affinity suggests that these avidin fusion proteins can be effectively used to deliver biotinylated ligands such as drugs and peptides to a certain locale, such as the brain.
Avidin ; Biotin* ; Brain ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Genetic Engineering ; Immunoglobulin G ; Ligands ; Molecular Weight ; Peptides ; Serum Albumin, Bovine

Plasmid vectors with either RSV or CMV promoter are frequently used for DNA- mediated immunization due to the availability in commercial. Consequently, influence of the vector constituents, such as promoter, enhancer and transcription termination signal etc. on vaccination efficiency is not studied extensively. As an initial attempt to develop an efficient vector system for DNA-rnediated immunization, influence of promoter for antigen gene expression on vaccination efficiency has been analyzed. Initially, plasmids with either B-actin or muscle creatine kinase (MCK) promoter were constructed from the plasmid with prototype CMV promoter. In addition, ovalbumin (OVA) antigen gene has been cloned into each vectors to generate the plasmid vectors with different promoters for induction of the anti-OVA immune responses. Antigen protein expression in antigen gene transfected mouse muscle myoblast cells showed that the level from MCK promoter containing plasmid was slightly higher than those from either CMV or B-actin promoter containing plasmids. Also, the same plasmid turned out to be slightly more efficient than other plasmids in antibody imrnune response induction in vivo, when they were applied both through intramuscularly and intradermally. These results suggest that the commonly used CMV promoter containing plasmid vector could be further modified to develop an efficient vector for DNA-mediated immunization.
Animals ; Clone Cells ; Creatine Kinase, MM Form ; Gene Expression ; Immunization ; Mice ; Myoblasts ; Ovalbumin ; Plasmids* ; Vaccination*

"Mig is a gamma interferon-inducible T cell chemoattractant that is a member of the chemokine family of cytokines. In order to gain a better understanding of the molecular mechanisms that regulate expression of the Mig gene, we have characterized the Mig gene and compared its structure and regulatory sequences with that of its ciosest IP10 gene. The genomic organization of the Mig gene reveals three introns that interrupt the transcribed sequence into four functional domains with a single ""CAT""- and ""TATA""-like structure. Primer extension analysis was used to identify the transcriptional initiation site that is located 50 bp upstream to the methionine codon that begins the long open reading frame. Comparison of the intron-exon structure of this gene to the gene for IP10 establishes that both genes are interrupted in precisely the same positions within homologous codons. The similarity of the intron-exon structure of the Mig and IP10 genes further support the hypothesis that Mig and IP10 genes have evolved from a common ancestral gene by gene duplication. The 5'-flanking region of Mig gene shows no overall sequence similarity with that from its closest IP10 gene whose production is also affected by gamma interferon. However, there are regions including a sequence with similarity to the NFxB binding site, AP-1 binding site, and ISRE. The r-RF-1 binding site is well conserved from -204 to -194 from the transcription start site in the Mig gene. Given the importance of IFN-r for effective immunity in tuberculosis and induction of Mig and IP10 genes in macrophages by IFN-r, we demonstrated induction of the genes Mig and IP10 with different message levels in the THP-1 human monocytic cell lines stimulated with whole M. tuberculosis. Despite the very similarity in genomic organization and the overlap in biological activities between MIG and IP10, our data described herein further support the suggestion that these chemokines rnay role nonredundantly in vivo. Moreover, our studies done on the Mig gene should provide the structural framework for future studies and begin to dissect cis-acting DNA sequences that are critical for gene regulation mediated by cell surface receptors."
Base Sequence ; Binding Sites ; Cell Line ; Chemokine CXCL9* ; Chemokines ; Codon ; Cytokines ; Gene Duplication ; Genome ; Humans* ; Interferons ; Introns ; Macrophages ; Methionine ; Open Reading Frames ; Transcription Factor AP-1 ; Transcription Initiation Site ; Tuberculosis

The effects of chimeric monoclonal antibodies (cMAbs), prostaglandin E, (PGE,), and indomethacin (INDO) on antibody-dependent cellular cytotoxicity (ADCC) against human squamous cell carcinoma of head and neck (SCCHN) cell line were examined. Using the PCI-50 SCCHN cell line as target and normal human peripheral blood mononuclear cells as effector, ADCC was enhanced by the treatment of cMAbs (1.25 p,g/ml), but was inhibited by exogenous PGE (5 X 10' M). The effects of cMAb and PGE were dose-dependent. Maximal suppression of activity occured when PGE was present during the entire 4-hr 'Cr-release assay period, whereas pretreatment of effector cells with PGE had minimal inhibitory effect after washing. These results indicate that decreased ADCC seen with SCCHN targets treated with PGE is related to post-binding events, such as binding of effector and target cells. Pre-treatment of effector cells with INDO (1 ug/ml) resulted in restoration of NK activity which was inhibited by PGE. Our in vitro results suggest that INDO can increase tumor cell killing by the reversal of the suppression for many imrnune functions by PGE.
Antibodies, Monoclonal ; Antibody-Dependent Cell Cytotoxicity ; Carcinoma, Squamous Cell* ; Cell Line ; Head* ; Homicide ; Humans* ; Indomethacin ; Neck* ; Prostaglandins E

The pleckstrin homology (PH) domain is a protein module of approximately 100 amino acids, that has been found in signaling molecules, including serinelthreonine kinase, GTPase-activating protein, phospholipase, and some cytoskeletal proteins. Although the specific function of PH domain has not been defined yet, it is believed that this domain is involved in the regulation of signal transduction pathway. The expression plasmids of human PLCg PH domains were constructed to see the roles of them in IL-6 signal transduction. When these expression plasmids are transfected into B9 cells, only N-terminal of PH domain inhibited IL-6-induced B9 cell proliferation. These results suggest that N-terminal of PH domain is critical for IL-6 signal transduction in B9 cells. To search the binding proteins associated PH domains of PLCy1 in B9 cells, Glutathione S-trnaferase (GST) fusion proteins containg PH domains were expressed in E. coli. Then, IL-6-dependent B9 cells were treated with 10 unit/ml IL-6 and the cell lysates were immunoprecipited with GST-PH doman fusion proteins. In vitro kinase assay of immune complex demonstrated that p38 (38 KDa) protein was coprecipitated with NC fusion protein, but IL-6 had no additional effect on it. When S-methaionine labelled cell lysates were used for immunoprecipitation, the same result was observed, conforming the association of p38 with NC motive of PH domain.
Amino Acids ; Antigen-Antibody Complex ; Carrier Proteins ; Cell Proliferation ; Cytoskeletal Proteins ; Glutathione ; GTPase-Activating Proteins ; Humans ; Hydrogen-Ion Concentration ; Immunoprecipitation ; Interleukin-6 ; Phospholipases ; Phosphotransferases ; Plasmids ; Signal Transduction