It has been known that the immunological functions against cancer cells were diminished, and these phenomena result from the inhibition of cell-mediated immunity by substance(s) secreted from cancer cells. It was also reported that the immunological functions decreased in patients with stomach cancer, which is the most frequent cnacer in Korean. However, the nature and function of the inhibitory factor(s) orignated from stomach cancer have not been identified. To elucidate effects of immuological inhibitory factor(s) secreted from cancer cells, SNU-1 (stomach cancer) and SW480 P109/R3P2 (colon cancer) were used in this study. Jurkat T cell line, an acute T cell leukemia, was pre-incubated with fractionated cancer cell culture supernatant for 3 days, then was stimulated with PMA, PWVanti-CD28 mAb or PMA/ionomycin for 8 hrs respectively. Fraction of SNU-1 (3 - 10 kDa) and above 10 kDa of SW480 P109/R3P2 inhibited the expression of IFN-r mRNA when Jurkat T cell was stimulated with PMA. However, there were no difference in IL-2 and IL-4 gene expression response to either PMA/anti-CD28 mAb or PMA/ionomycin. These results show that cancer cells secret some inhibitory factor(s) acting on the immune response, especially IFN-r gene expression of the Jurkat T cells response to PMA. Therefore, it suggests that the inhibitory factor(s) secreted from cancer cells influences on. the PKC-dependent pathway related to the signal transduction by PMA.
Cell Culture Techniques ; Cell Line ; Gene Expression ; Humans ; Immunity, Cellular ; Interleukin-2 ; Interleukin-4 ; Leukemia, T-Cell ; RNA, Messenger ; Signal Transduction ; Stomach Neoplasms ; T-Lymphocytes

HLA-A2 is present at high frequency in most populations, as identified by serological and biochemical means. The values of these methods are limited by their failures to discriminate the products of the known allelic HLA-A02 variants. The great majority of genetic polymorphism which defines the allelic variants is found in exons 2 and 3 of the HLA-A02 gene. These exons encode the a-1 and a-2 domains of the HLA class I molecules, and the variation within the genes may influence the peptide binding specificity of the gene products of each allele. To determine the 17 known alleles of HLA-A02 an ARMS-PCR was developed. We applied this ARMS-PCR to 10 standard cell lines and we confirmed the specificity and sensitivity of this method. We defined the HLA-A 02 subtypes in 146 healthy Koreans who were serologically identified as HLA-A2. Five subtypes out of the 17 known A02 alleles were detected (A'0201, 0203, 0206, 0207, '0210) and A'0201 was most frequent (53.4%) and A'0206, '0207, '0203, 0210 (37.0%, 18.5%, 2.7%, 2.1%), were followed respectively. By linkage disequilibrium analysis with HLA-B alleles, A*02 subtypes were defined to be associated with many B alleles (B27, 35, 38, 39, 46, 52, 60, and 61). It is suggested that these findings may be helpful for the selection of patients for the specific immunotherapy with HLA-A02 restricted peptide vaccines and for the unrelated bone marrow transplantation in Korean.
Alleles ; Bone Marrow Transplantation ; Cell Line ; Exons ; HLA-A Antigens ; HLA-A2 Antigen ; HLA-B Antigens ; Humans ; Immunotherapy ; Linkage Disequilibrium ; Polymorphism, Genetic ; Sensitivity and Specificity ; Vaccines, Subunit

Normal human B cells produce autocrine growth factor in response to Staphylococcus aureus Cowan I strain (SAC). However, the functional role and molecular nature of the B cell derived-B cell growth factor (B-BCGF) are largely unknown. We have tried to investigate the nature of B-BCGF using mAb for several years. We report here that B- BCGF is capable of binding to hemoglobin (Hb). The concentrated culture supernatant from tonsillar B cells stimulated with SAC for 24 h was loaded into the fast protein liquid chromatography and ion-exchange chromatography. The peak with BCGF activity was shown to have a M.W. of 16-18 Kda in polyacrylamide gel electrophoresis followed by silver stain. Amino acid sequence of the fraction was found to identical to human hemoglobin (Hb) by more than 85%. However, Hb itself had no BCGF activity. The presence of Hb in culture supernatant was due to the contamination of SRBC during B cell purification. SRSC were completely removed from B cells by percoll-gradient centrifugation and B cells were stimulated with SAC and exogenous Hb was added to the cultures. The Hb fraction from FPLC again showed a BCGF activity. These data strongly suggested that BCGF binds to Hb. We confirmed this in dot blot as well as Western blot. The M.W of Hb-binding BCGF was 20 Kda. This information may provide a rapid progress in research of BCGF.
Amino Acid Sequence ; B-Lymphocytes ; Blotting, Western ; Centrifugation ; Chromatography, Ion Exchange ; Chromatography, Liquid ; Electrophoresis, Polyacrylamide Gel ; Humans ; Silver ; Staphylococcus aureus

Bipotent progenitors for T and natural killer (NK) lymphocytes are thought to exist among early precursor thymocytes or liver lymphocytes. The identification of such a progenitor population or mature NK cells in such organs remains undefined. Here we report the identification of a novel receptor of NK cells, p58 (HLA class I-specific inhibitory receptors), in fetal thymocytes and fetal liver lymphocytes. Our finding suggests the NK cells mature in the developmental stage during feta1 ontogeny. Flow cytometric analysis revealed p58 positive cells in thymocytes or in fetal liver lymphocytes and reverse transcription PCR also showed amplification of p58 RNA. The result of single stranded conformational polymorphism (SSCP) showed it discriminates one or two base pair differences of the p58 gene. Although the question still remains as to whether the expression of p58 is due to the NK cells or natural T cells, it is clear the p58 is expressed in fetal thymocytes or liver lymphocytes. And SSCP analysis using appropriate sets of primers used in this study, is helpful to study the diversity of p58.
Base Pairing ; Killer Cells, Natural ; Liver* ; Lymphocytes* ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Reverse Transcription ; RNA ; T-Lymphocytes ; Thymocytes*

The gag encoded p24 protein of human immunodeficiency virus-1 (HIV-1) is a major constitutent of the viral core, and is also known as one of the most immunodominant antigens in the host immune response against the HIV-1. Based on the neutralizing ability of anti-p24 antibodies as well as their rapid appearance in human serum after viral infection, the development of vaccines and diagnostic tools targeting the p24 protein and anti-p24 antibodies is of great interest. For the characterization of the immunological properties of the HIV-1 p24 protein, in a previous study, putative B-cell epitopes were identified by screening the reactivity of a goat anti-p24 antiserum to a large array of overlapping synthetic peptides covering the whole p24 sequence. Four peptides were identified for their abilities to elicit a strong B-cell response, which sequences comprises the regions p24 (164-182), (202-221), (217-236) and (232-256), respectively. In the present study, the immunogenicity and differential properties of each of these individual epitopes were further characterized. To evaluate the time course of the antibody response, BALB/c mice were immunized with the HIV-1 p24 protein and their serum titers against each of these peptides were determined. The earliest immune response was observed against the p24 (202-221) peptide, which also showed the highest antibody titer against the immunized antigen. Furthermore,. enzyme-linked immunosorbent assay with HIV-1 p24 protein coated microtiter plates revealed that anti-p24 (202-221) antiserum has the most pronounced reactivity against the native p24 protein. Since the p24 (202-221) epitope has also been reported to include a cytotoxic T-lymphocyte epitope, it is suggested that this region might represent a powerful antigenic site responsible for eliciting both T- and B-cell immune response. The possible application of this specific epitope in vaccine development or AIDS diagnosis is discussed.
Animals ; Antibodies ; Antibody Formation ; B-Lymphocytes ; Diagnosis ; Enzyme-Linked Immunosorbent Assay ; Epitopes* ; Epitopes, B-Lymphocyte ; Goats ; HIV* ; HIV-1 ; Humans ; Immunodominant Epitopes ; Mass Screening ; Mice ; Peptides* ; T-Lymphocytes, Cytotoxic ; Vaccines

Transforming growth factor-j31 (TGF-p1) has potential for therapeutic use in common clinical conditions for which there are no adequate pharmacological agents. However, in vivo studies using TGF-p1 were hindered by high price of this cytokine. As a first step towards large scale purification of TGF-p1, it was purified in a small scale (10 unit platelets) from human platelets by four purification steps: platelet extraction, gel filtration, cation exchange chromatography, and reversed phase high performance liquid chromatography (HPLC). A single protein band with a molecular weight of 25 Kd corresponding to purchased TGF-p1 (R8D Systems) was confirmed by silver staining after SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of eluant from reversed phase HPLC. Recovery (%) of each step was about 50-60%, resulting in the final recovery of 20% based on the detection by a sandwich ELISA. Approximately, 3.7 p,g of purified TGF-p1 was obtained from 18 pg of platelet extracts. This result was confirmed by receptor (TGF-j31 type II) ELISA and bioassay using a mink lung epithelial'cell line (MV1LU). Further, in vitro characterization study showed that purified TGF-p1 inhibits G1/S transition of LPS-activated murine spleen B cells and increases surface IgA expression by the same cell population, which are typical activities of TGF-p1 in B cell differentiation. Taken together, the results from the present study reveals that purified TGF-p1 is fully biologically active and our purification methodology could be usbful to obtain a large scale of recombinant TGF-p1 in the future.
B-Lymphocytes ; Biological Assay ; Blood Platelets ; Cell Cycle ; Cell Differentiation ; Chromatography ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Electrophoresis ; Enzyme-Linked Immunosorbent Assay ; Humans* ; Immunoglobulin A ; Lung ; Mink ; Molecular Weight ; Silver Staining ; Spleen ; Transforming Growth Factors*

GTPrS-dependent phospholipase D activity in human neutrophils was investigated using exogenous phospholipid as a substrate. Both cytosolic and membrane- associated phospholipase D activities were identified. The previously described 50 kDa cytosolic activating factor was resolved chromatographically from the cytosolic phospholipase D. Using exogenous phospholipid as substrate along with chromatographically resolved 50 kDa factor and recombinant ADP-ribosylation factor 1, plasma membrane was required for activity, indicating that the activity which was previously seen using endogenous phospholipid substrate was due to a phospholipase D located in the plasma membrane. In addition, ADP-ribosylation factor and the 50 kDa factor activated synergistically. Using neutrophil plasma membranes, a third regulator of neutrophil membrane phospholipase D was identified from bovine brain cytosol. This factor was resolved from ADP-ribosylation factor and Rho A by successive column chromatographies. The brain factor showed a synergistic effect with the 50 kDa neutrophil activator but an additive effect with recombinant ADP- ribosylation factor. Whether or not ADP-ribosylation factor or the brain factor were present, high activities were seen only when the 50 kDa factor was present, indicating that the 50 kDa cytosolic factor is a major activating factor for the neutrophil plasma membrane phospholipase D.
ADP-Ribosylation Factor 1 ; ADP-Ribosylation Factors* ; Brain* ; Cell Membrane ; Chromatography ; Cytosol* ; Fibrinogen ; Humans ; Membranes ; Neutrophils* ; Phospholipase D* ; Phospholipases*

No abstract available.
Brain* ; Nitrogen* ; Oxygen*

No abstract available.
Animals ; Antigen-Antibody Complex* ; Bacteria* ; Mice*

No abstract available.
Escherichia coli* ; Escherichia*