Immunoadjuvant activities of the extract (KM-110) from Korean mistletoe ( Viscum album coloratum) on the induction of humoral and cellular responses against Keyhole limpet hemocyanim (KLH) as an antigen and allogenic tumor cells were examined. When mice were immunized subcataneously (s.c.) with KLH admixed with KM-110, more than 1000-times higher antibody titers to KLH than those immunized with KLH alone was observed. KM-110 induced high level of KLH- specific IgG1, IgG2a and IgG2b antibodies. In an in vitro analysis of lymphocytes proliferation to KLH on week 4, the splenocytes of mice treated with KLH and KM- 110 exhibited significantly higher proliferating activity than those treated with KLH alone. In addition, the culture supernatnats obtained from the splenocytes of mice treated with both KLH and KM-110 showed high level of IL-2 and IL-4. In the test of cellular immune responses, KM-110 enhanced the DTH reaction to KLH in mice. Furthemore, cytotoxic T-lymphocyte (CTL) activity using an allogenic CTL induction model where C57BL/6 (H-2b) mice were injected with allogenic P815 (H-2d) mastocytoma cells admixed with or without KM-110, mice treated with P815 cells and KM-110 showed higher cytatoxic activity against allogenic tumor cells than those treated without KM-110. This results suggest that KM-110 may possess adjuvant activities to potentially enhance humoral as well as cellular immune responses against antigens.
Animals ; Antibodies ; B-Lymphocytes ; Immunity, Cellular* ; Immunoglobulin G ; Interleukin-2 ; Interleukin-4 ; Lymphocytes ; Mastocytoma ; Mice ; Mistletoe* ; T-Lymphocytes ; T-Lymphocytes, Cytotoxic ; Viscum album* ; Viscum*

Immunological effect of the extract (KM-110) from Korean mistletoe (Viscum album coloratum) was examined. Lymphocytes obtained from KM-110-administrated mice showed increased responsiveness to mitogens, concanavalin-A (Con.A) and lipopolysaccharide (LPS). In order to study cytokine induct ability of the KM-110, macrophages from the Balb/c mice were cultivated in the medium containing the extract. the macrophages were shown to induce secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin 1 and 6 (IL-1, IL-6) and interferon-gamma (INF-gamma). We then tested antitumor activity of the macrophages activated by the KM-110. Peritoneal macrophages harvested from the KM110-treated Balb/c mice exhibited cytotoxicity against the syngeneic 3LL carcinoma cells. ...continue...
Animals ; Interferon-gamma ; Interleukin-1 ; Lymphocytes ; Macrophages ; Macrophages, Peritoneal ; Mice ; Mistletoe* ; Mitogens ; Tumor Necrosis Factor-alpha ; Viscum album* ; Viscum*

Mycobacterium tuberculosis is a potent inducer of cytokine production by mononuclear phagocytes, which are an important cellular component in the first line immune defence. In this study, the cell wall-associated Triton X-100 soluble protein (TSP) antigens, TSP-H37Rv, TSP-H37Ra, TSP-K, and TSP-BCG, were isolated from M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. tuberculosis K-strain, and M. bovis BCG, respectively. The monocytes were isolated from the peripheral blood mononuclear cells of healthy individuals and were co-cultured with each TSP antigens and the secretory proteins of M. tuberculosis (PPD and 30-kDa antigen) to measure the production of cytokines; tumor necrosis factor (TNF)-a, interleukin (IL)-12, IL-8 and monocyte chemotactic protein-1 (MCP-1). The TSP-H37Rv antigen- stimulated monocytes showed higher level of TNF-a and IL-12 production compared to those of other TSP antigens and PPD. Especially, IL-12 production in response to the TSP-H37Rv antigen was significantly elevated in comparison with that of PPD-stimulated monocytes (TSP-H37Rv, 255.5+/-256.9 pg/ml; PPD, 55.7+/-55.4 pg/ml). However, the 30-kDa antigen did not induce TNF-alpha expression and also showed the lowest level of cytokine and chemokine production by monocytes. MCP-1 and IL-8 production were similarly increased in response to all TSP antigens and the PPD antigen. The production of IL-12 by the TSP-H37Rv antigen stimulation was significantly increased in PPD reactors than that in the non-reactor group, while the levels of other cytokines stimulated with each TSP antigens, 30-kDa and PPD antigen were not significantly different between the tuberculin reactor and the non-reactor groups. These results suggest that the cell wall-associated TSP antigen isolated from M. tuberculosis H37Rv acts as a more potent IL-12 inducer than the PPD antigen in innate immune response and thus it could further activate the Th1-mediated immune responses effectively against M. tuberculosis infection.
Chemokine CCL2 ; Cytokines ; Humans* ; Immunity, Innate ; Interleukin-10* ; Interleukin-12* ; Interleukin-8* ; Interleukins ; Monocytes* ; Mycobacterium bovis ; Mycobacterium tuberculosis* ; Mycobacterium* ; Neptune* ; Octoxynol* ; Phagocytes ; Tuberculin ; Tuberculosis ; Tumor Necrosis Factor-alpha*

Tremendous efforts have been made to develop better vaccines and diagnostic markers for the effective control of tuberculosis. Recently, we reported that the Triton X-100 soluble protein (TSP) of Mycobacterium tuberculosis induced strong T-cell proliferation and IFN-gamma production in humans, and also conferred a significant level of protection against tuberculosis in a mouse model. In this study, the TSP was prepared by Triton X-100 extraction of Mycobacterium tuberculosis bacilli, which was followed by Triton X-114 phase partitioning. Western blot analysis using sera of 177 active pulmonary tuberculosis patients and 323 healthy individuals revealed that the TSP contained a immunodominant 40-kDa antigen specifically reacting with some sera from pulmonary tuberculosis patients. The 40-kDa antigen was purified by ion-exchange chromatography, and partially characterized by two-dimensional gel electrophoresis and N-terminal sequencing. Results of this study suggest that 40-kDa molecule of the TSP antigen from the cell suface of Mycobacterium tuberculosis can be used as a serodiagnostic marker as well as a potential vaccine candidate against tuberculosis.
Animals ; Blotting, Western ; Chromatography, Ion Exchange ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Mice ; Mycobacterium tuberculosis* ; Mycobacterium* ; Neptune* ; Octoxynol* ; T-Lymphocytes ; Tuberculosis ; Tuberculosis, Pulmonary ; Vaccines

Dectin-1, which specifically recognizes beta-glucan of fungal cell walls, is a non-Toll-like receptor (TLR) pattern recognition receptor and a representative of C-type lectin receptors (CLRs). The importance of Dectin-1 in innate immune cells, such as dendritic cells and macrophages, has previously been well studied. However, the function of Dectin-1 in B cells is very poorly understood. To determine the role of Dectin-1 in B cell activation, we first investigated whether mouse B cells express Dectin-1 and then assessed the effect of Dectin-1 stimulation on B cell proliferation and antibody production. Mouse B cells express mRNAs encoding CLRs, including Dectin-1, and surface Dectin-1 was expressed in B cells of C57BL/6 rather than BALB/c strain. Dectin-1 agonists, heat-killed Candida albicans (HKCA) and heat-killed Saccharomyces cerevisiae (HKSC), alone induced B cell proliferation but not antibody production. Interestingly, HKSC, HKCA, and depleted zymosan (a selective Dectin-1 agonist) selectively enhanced LPS-driven IgG1 production. Taken together, these results suggest that, during fungal infection, beta-glucan-stimulated Dectin-1 may cooperate with TLR4 to specifically enhance IgG1 production by mouse B cells.
Animals ; Antibody Formation ; B-Lymphocytes* ; Candida albicans ; Cell Proliferation ; Cell Wall ; Dendritic Cells ; Immunoglobulin G* ; Lectins, C-Type ; Macrophages ; Mice* ; RNA, Messenger ; Saccharomyces cerevisiae ; Sprains and Strains ; Zymosan

To identify new bio-markers as well as potential targets for new drugs for epithelial ovarian cancer (EOC), we compared the gene expression profiles of cancer tissues from 25 EOCs with human ovarian surface epithelium (HOSE) using in-house cDNA microarray specified to EOC. Based on a comprehensive method and information from the National Cancer Institute (NCI) Cancer Genome Anatomy Project (CGAP), the cDNA library was constructed. After excluding the overlapping clones, 768 spots were included in the array. We identified the genes and expressed sequence tags (ESTs) (30 up-regulated and 34 down-regulated) that are differentially expressed in EOC tissues. To confirm the expression data, we performed real time RT-PCR experiments. Using microdissected EOC tissues and cell lines, we investigated the expression status of the NET-1 gene, clusterin gene, and actin-binding LIM protein 1. The information provided here will be useful for identifying genes whose products might serve as molecular signatures for the biomakers of EOCs.
Cell Line ; Clone Cells ; Clusterin ; DNA, Complementary* ; Epithelium ; Expressed Sequence Tags ; Gene Expression* ; Gene Library ; Genome ; Humans ; National Cancer Institute (U.S.) ; Oligonucleotide Array Sequence Analysis* ; Ovarian Neoplasms ; Transcriptome*

Immune cells express toll-like receptors (TLRs) and respond to molecular patterns of various pathogens. CpG motif in bacterial DNA activates innate and acquired immune systems through binding to TLR9 of immune cells. Several studies reported that CpG can directly regulate B cell activation, differentiation, and Ig production. However, the role of CpG in B cell growth and Ig production is not fully understood. In this study, we analyzed the effect of CpG on the kinetics of mouse B cell viability, proliferation, and Igs production. Overall, CpG enhanced mouse B cell growth and production of Igs in a dose-dependent manner. Unlike LPS, 100 nM CpG (high dose) did not support TGF-beta1-induced IgA and IgG2b production. Moreover, 100 nM CpG treatment abrogated either LPS-induced IgM or LPS/TGF-beta1-induced IgA and IgG2b production, although B cell growth was enhanced by CpG under the same culture conditions. We subsequently found that 10 nM CpG (low dose) is sufficient for B cell growth. Again, 10 nM CpG did not support TGF-beta1-induced IgA production but, interestingly enough, supported RA-induced IgA production. Further, 10 nM CpG, unlike 100 nM, neither abrogated the LPS/TGF-beta1-nor the LPS/RA-induced IgA production. Taken together, these results suggest that dose of CpG is critical in B cell growth and Igs production and the optimal dose of CpG cooperates with LPS in B cell activation and differentiation toward Igs production.
Animals ; Cell Survival ; DNA, Bacterial ; Immune System ; Immunoglobulin A ; Immunoglobulin G ; Immunoglobulin M ; Immunoglobulins ; Kinetics ; Mice ; Toll-Like Receptors

The purification of immunodominant native protein antigens from the culture filtrates of Mycobacterium tuberculosis is needed for the development of new vaccines and immunodiagnostic reagents against tuberculosis. In the present study, we conducted large scale purification of well-known secreted antigens, Ag85 complex, 38-kDa, and MTB12, from the culture filtrate proteins (CFPs) prepared from M. tuberculosis H37Rv grown as a surface pellicle on synthetic Sauton medium. The protein and antigen concentrations of culture filtrates were sufficiently increased after 6 week of culture. The MTB12 antigen was detected as early as 1 week of culture, and Ag85 complex and 38-kDa antigen were detected after 2 and 3 week of culture, respectively, by immunodiffusion with specific antiserum against 100-fold concentrated culture filtrates. For large-scale purification, the six-week-culture filtrates of M. tuberculosis H37Rv diluted 2.5-fold with 20 mM Tris-HCl, (P)H 8.3 were subjected to anion-exchange chromatography. The CFPs were eluted with 100 mM NaCl-20 mM Tris-HCl, pH 8.3 and concentrated by ultrafiltration. The concentrated CFPs were fractionated with ammonium sulfate, and followed by hydrophobic interaction chromatography and anion-exchange chromatography (FPLC). Eventually, 10 mg of Ag85 complex, 0.56 mg of 38-kDa, and 1.81 mg of MTB12 antigens were purified from 1 liter of the six-week-culture filtrates of M. tuberculosis H37Rv which contained 307.81 mg of protein of culture filtrate.
Ammonium Sulfate ; Chromatography ; Hydrogen-Ion Concentration ; Hydrophobic and Hydrophilic Interactions ; Immunodiffusion ; Indicators and Reagents ; Mycobacterium tuberculosis* ; Mycobacterium* ; Tuberculosis ; Ultrafiltration ; Vaccines

TGF-beta induces IgA class switching by B cells. We previously reported that Smad3 and Smad4, pivotal TGF-beta signal-transducing transcription factors, mediate germline (GL) alpha transcription induced by TGF-beta1, resulting in IgA switching by mouse B cells. Post-translational sumoylation of Smad3 and Smad4 regulates TGF-beta-induced transcriptional activation in certain cell types. In the present study, we investigated the effect of sumoylation on TGF-beta1-induced, Smad3/4-mediated GLalpha transcription and IgA switching by mouse B cell line, CH12F3-2A. Overexpression of small ubiquitin-like modifier (SUMO)-1, SUMO-2 or SUMO-3 did not affect TGF-beta1-induced, Smad3/4-mediated GLalpha promoter activity, expression of endogenous GLalpha transcripts, surface IgA expression, and IgA production. Next, we tested the effect of the E3 ligase PIASy on TGF-beta1-induced, Smad3/4-mediated GLalpha promoter activity. We found that PIASy overexpression suppresses the GLalpha promoter activity in cooperation with histone deacetylase 1. Taken together, these results suggest that SUMO itself does not affect regulation of GLalpha transcription and IgA switching induced by TGF-beta1/Smad3/4, while PIASy acts as a repressor.
Animals ; B-Lymphocytes* ; Cell Line ; Histone Deacetylase 1 ; Immunoglobulin A ; Immunoglobulin Class Switching ; Mice ; Small Ubiquitin-Related Modifier Proteins* ; SUMO-1 Protein* ; Sumoylation ; Transcription Factors ; Transcriptional Activation ; Transforming Growth Factor beta ; Transforming Growth Factor beta1 ; Ubiquitin-Protein Ligases

Ginsenosides are the major components of ginseng, which is known to modulate blood pressure, metabolism, and immune function, and has been used to treat various diseases. It has been reported that ginseng and several ginsenosides have immunoregulatory effects on the innate and T cell-mediated immune response. However, their effects on the humoral immune response have not been fully explored. The present study examined the direct effects of red ginseng extract (RGE) and ginsenosides on mouse B cell proliferation and on antibody production and the expression of germline transcripts (GLT) by mouse B cells in vitro. RGE slightly reduced B cell proliferation, but increased IgA production by LPS-stimulated B cells. Furthermore, ginsenoside Rg1 and 20(S)-Rg3 selectively induced IgA production and expression of GLTalpha transcripts by LPS-stimulated B cells. Collectively, these results suggest that ginsenoside Rg1 and 20(S)-Rg3 can drive the differentiation of B cells into IgA-producing cells through the selective induction of GLTalpha expression.
Animals ; Antibody Formation ; B-Lymphocytes* ; Blood Pressure ; Cell Proliferation ; Ginsenosides ; Immunity, Humoral ; Immunoglobulin A* ; Metabolism ; Mice* ; Panax