1.Regulation of transforming growth factor-beta1 synthesis by nurine T lymphocytes and its effect of IgA antibody response.
Kyoung Bum SEO ; Se Won YIE ; Inpyo CHOI ; Kwang Ho PYUN ; Pyeung Hyeun KIM
Korean Journal of Immunology 1992;14(2):203-211
No abstract available.
Antibody Formation*
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Immunoglobulin A*
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T-Lymphocytes*
2.Characterization of Mouse B Lymphoma Cells (CH12F3-2A) for the Study of IgA Isotype Switching.
Young Saeng JANG ; Seo Hyeun CHOI ; Seok Rae PARK ; Hyun A KIM ; Jae Bong PARK ; Pyeung Hyeun KIM
Immune Network 2004;4(4):216-223
BACKGROUND: It is well known that IgA isotype switching is induced by TGF-beta1. LPS-activated mouse normal B cells well differentiate into IgA secreting plasma cells under the influence of TGF-beta1. Nevertheless, there are lots of difficulties in studying normal B cells in detail because it is not simple to obtain highly purified B cells, showing low reproducibility and transfection efficacy, moreover impossible to keep continuous culture. To overcome these obstacles, it is desperately needed to develop B cell line which acts like normal B cells. In the present study, we investigated whether CH12F3-2A lymphoma cells are appropriate for studying IgA isotype switching event. METHODS: CH12F3-2A B cell line was treated with LPS and TGF-beta1, then levels of germ-line (GL) transcripts were measured by RT-PCR, and GLalpha promoter activity was measured by luciferase assay. In addition, membrane IgA (mIgA) expression and IgA secretion were determined by FACS and ELISA, respectively. RESULTS: TGF-beta1, regardless of the presence of LPS, increased level of GLalpha transcripts but not GLgamma2b transcripts. However, IgA secretion was increased dramatically by co-stimulation of LPS and TGF-beta1. Both mIgA and IgA secretion in the presence of TGF-beta1 were further increased by over-expression of Smad3/4. Finally, GLalpha promoter activity was increased by TGF-beta1. CONCLUSION: CH12F3-2A cell line acts quite similarly to the normal B cells which have been previously reported regarding IgA expression. Thus, CH12F3-2A lymphoma cell line appears to be adequate for the investigation of the mechanism(s) of IgA isotype switching at the cellular and molecular levels.
Animals
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B-Lymphocytes
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Cell Line
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Enzyme-Linked Immunosorbent Assay
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Immunoglobulin A*
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Immunoglobulin Class Switching*
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Luciferases
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Lymphoma*
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Membranes
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Mice*
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Plasma Cells
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Transfection
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Transforming Growth Factor beta1
3.Combined blockade of HER2 and VEGF exerts greater growth inhibition of HER2-overexpressing gastric cancer xenografts than individual blockade.
Rohit SINGH ; Woo Jin KIM ; Pyeung Hyeun KIM ; Hyo Jeong HONG
Experimental & Molecular Medicine 2013;45(11):e52-
Gastric cancer overexpressing the human epidermal growth factor 2 (HER2) protein has a poor outcome, although a combination of chemotherapy and the anti-HER2 antibody trastuzumab has been approved for the treatment of advanced gastric cancer. Vascular endothelial growth factor (VEGF) expression in gastric cancer is correlated with recurrence and poor prognosis; however, the anti-VEGF antibody bevacizumab has shown limited efficacy against gastric cancer in clinical trials. In this study, we evaluated the antitumor effects of trastuzumab; VEGF-Trap binding to VEGF-A, VEGF-B and placental growth factor (PlGF); and a combination of trastuzumab and VEGF-Trap in a gastric cancer xenograft model. Although trastuzumab and VEGF-Trap each moderately inhibited tumor growth, the combination of these agents exerted greater inhibition compared with either agent alone. Immunohistochemical analyses indicated that the reduction in tumor growth was associated with decreased proliferation and increased apoptosis of tumor cells and decreased tumor vascular density. The combined treatment resulted in fewer proliferating tumor cells, more apoptotic cells and reduced tumor vascular density compared with treatment with trastuzumab or VEGF-Trap alone, indicating that trastuzumab and VEGF-Trap had additive inhibitory effects on the tumor growth and angiogenesis of the gastric cancer xenografts. These data suggest that trastuzumab in combination with VEGF-Trap may represent an effective approach to treating HER2-overexpressing gastric cancer.
Animals
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Antibodies, Monoclonal, Humanized/administration & dosage/*therapeutic use
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Antineoplastic Combined Chemotherapy Protocols/*therapeutic use
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Apoptosis
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Cell Line, Tumor
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Cell Proliferation
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Humans
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Mice
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Mice, Inbred BALB C
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Neovascularization, Pathologic/drug therapy
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Receptor, erbB-2/*antagonists & inhibitors
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Receptors, Vascular Endothelial Growth Factor/administration & dosage/*therapeutic use
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Recombinant Fusion Proteins/administration & dosage/*therapeutic use
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Stomach Neoplasms/*drug therapy/pathology
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Vascular Endothelial Growth Factor A/antagonists & inhibitors
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Xenograft Model Antitumor Assays
4.Preferential Expression of IgA Isotype Switching-associated Transcripts in Mouse Intestinal Lymphoid Tissues.
Byung Chul CHAE ; Sung Ki CHUN ; Goo Young SEO ; Hyun A KIM ; Pyeung Hyeun KIM
Immune Network 2005;5(4):215-220
BACKGROUND: Transforming growth factor-beta1 (TGF-beta1) directs class switch recombination (CSR) to IgA isotype, which is a predominant antibody in mucosal surfaces. Although IgA is preferentially committed in mucosal lymphoid tissues, it is not definitely established whether hallmarks of IgA CSR such as IgA germ-line transcripts (GLTalpha), post-switch transcripts (PSTalpha) and circle transcripts (CTalpha) are readily expressed in such tissues. Therefore, we compared the expression of these transcripts among mouse Peyer's patches (PP), mesenteric lymph nodes (MLN), and spleen. METHODS: Levels of GLTs, PSTs and CTs were measured by RT-PCR in isolated PPs, MLNs and spleen cells. RESULTS: GLTalpha and PSTalpha were well expressed in PP and MLN cells but in spleen cells. Similar patterns were observed in the expression of GLgamma2b and PSTgamma2b. On the other hand, these transcripts were only inducible in spleen cells upon stimulated with LPS and TGF-beta1. In addition, CTalpha and CTgamma2b were detected in PP cells. CONCLUSION: PP B cells readily express IgA GLT, PST, and CT. Overall expression patterns of these transcripts were similar in MLN cells. Thus, these results suggest that microenvironment of PP and MLN influences spontaneous IgA CSR, which lacks in systemic lymphoid tissues such as spleen.
Animals
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B-Lymphocytes
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Hand
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Immunoglobulin A*
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Lymph Nodes
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Lymphoid Tissue*
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Mice*
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Peyer's Patches
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Recombination, Genetic
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Spleen
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Transforming Growth Factor beta1
5.Activin A Stimulates Mouse APCs to Express BAFF via ALK4-Smad3 Pathway.
Jae Hee KIM ; Goo Young SEO ; Pyeung Hyeun KIM
Immune Network 2011;11(4):196-202
BACKGROUND: B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells, and stimulates B cell proliferation, differentiation, survival, and Ig production. In the present study, we explored the effect of activin A on BAFF expression by APCs. METHODS: To investigate the effect of activin A on BAFF expression by mouse APCs, we measured the level of BAFF expression at the transcriptional and protein levels using RT-PCR and ELISA. RESULTS: Activin A markedly enhanced BAFF expression in mouse macrophages and dendritic cells at both the transcriptional and protein levels. SB431542, an activin receptor-like kinase 4 (ALK4) inhibitor, completely abrogated activin A-induced BAFF transcription. Furthermore, overexpression of DN-Smad3 abolished activin-induced BAFF expression at the transcriptional and protein levels. CONCLUSION: These results demonstrate that activin A can enhance BAFF expression through ALK4-Smad3 pathway.
Activin Receptors
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Activins
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Animals
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Benzamides
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Cell Proliferation
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Dendritic Cells
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Dioxoles
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Humans
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Macrophages
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Mice
6.Further Characterization of Activin A-induced IgA Response in Murine B Lymphocytes.
Hwa Joung LEE ; Pyeung Hyeun KIM
Immune Network 2009;9(4):133-137
We have recently shown that activin A, a member of TGF-beta superfamily, stimulates mouse B cells to express IgA isotype but other isotypes. In the present study, we further characterized effects of activin A on B cell growth and IgA expression. We found that activin A did not have effect on LPS-stimulated cell viability. In parallel, CFSE staining analysis revealed that activin A did not alter cell division. An increase of IgA secretion by activin A was completely abrogated by anti-activin A Ab but not by anti-TGFbeta1 Ab. In the same conditions, no other isotypes are significantly affected by each antibody treatment. Finally, activin A, as similar to TGF-beta1, increased IgA secretion by mesenteric lymph node cells. These results suggest that activin A can specifically stimulate IgA response, independent of TGF-beta in the gut.
Activins
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Animals
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B-Lymphocytes
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Cell Division
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Cell Survival
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Fluoresceins
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Immunoglobulin A
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Lymph Nodes
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Mice
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Succinimides
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Transforming Growth Factor beta
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Transforming Growth Factor beta1
7.TGF-beta1 induces mouse dendritic cells to express VEGF and its receptor (Flt-1) under hypoxic conditions.
Eun Hee NAM ; Seok Rae PARK ; Pyeung Hyeun KIM
Experimental & Molecular Medicine 2010;42(9):606-613
Angiogenesis is a multi-step process that involves the activation, proliferation, and migration of endothelial cells. We have recently shown that TGF-beta1 can induce mouse macrophages to produce VEGF, a potent angiogenic factor. In the present study, we explored whether TGF-beta1 has a similar effect on mouse dendritic cells. First, we show that under hypoxic conditions, TGF-beta1 induced the expression of VEGF transcripts in bone marrow-derived dendritic cells. Overexpression of Smad3/4 further augmented TGF-beta1-induced VEGF transcription, while overexpression of DN-Smad3 decreased VEGF transcription in DC2.4 cells, a mouse dendritic cell line. We also show that TGF-beta1 and Smads are involved in the induction of VEGF protein secretion. Interestingly, under the same conditions, the expression of VEGF receptor 1 (Flt-1) was also elevated at both the transcriptional and protein levels. Additionally, we found that the TGF-beta1-induced VEGF secretion in activated DC2.4 cells has wound-healing properties. Finally, Smad7 and Smurf1 negatively regulated the TGF-beta1-induced and Smad3/4-mediated VEGF expression. Taken together, these results indicate that TGF-beta1 can enhance the expression of VEGF and Flt-1 through the typical Smad pathway in mouse dendritic cells.
Angiogenesis Inhibitors/*metabolism
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Animals
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Cell Line
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Dendritic Cells/*metabolism
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Macrophages/metabolism
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Mice
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Mice, Inbred BALB C
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RNA, Messenger/metabolism
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Signal Transduction
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Smad2 Protein/metabolism
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Smad3 Protein/metabolism
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Smad4 Protein/metabolism
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Smad7 Protein/metabolism
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Transforming Growth Factor beta1/metabolism/*pharmacology
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Vascular Endothelial Growth Factor A/*secretion
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Vascular Endothelial Growth Factor Receptor-1/*metabolism
8.In vitro and In vivo Characterization of EL4 Cell Transfected with TGF-B1 cDNA.
Jung Hee LEE ; Jin Su YOO ; Ki Jong RHEE ; Min Hyoung SONG ; Won Kyo HAN ; Min Chul LEE ; Pyeung Hyeun KIM
Korean Journal of Immunology 1998;20(2):101-108
Transforming growth factor-B1 (TGF-B1) is well known to be one of the most potent Immunosuppressive cytokines. To determine whether TGF-B1 secreted in the latent form can be immunoregulatory, TGF-B1 cDNA driven by the human -actin promotor was transfected into a murine thymoma cell line, EL4 cells. The transfectants (ELJ4) secreted a latent torm of TGF-B1 at a concentration of 5 ng/ml under the influence of TPA. Transfected TGF-b1 transcripts was readily detected by RT-PCR in ELJ4 cells regardless of the presence of TPA, but not in EL4 cells. In addition, we found the degree of Thy-B1 expression, IL-2 secretion and the proliferation rate are not altered by the transfection. Finally, EL4 and ELJ4 cells were injected into C57BU6 mice (syngenic strain), subcutaneously. Tumor cell masses derived from both cell populations survived longer than 1 wk, and the size of tumor derived from ELJ4 was three times larger (2.5 cm of diameter) than that from EL4. Virtually, there was no histopathological difference between two tumors. Taken together, the results from the present study indicates that EL4 thymomas transfected with TGF-1 secretes a latent form of TGF-B1 which may suppress host immune defence system.
Humans
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Mice
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Animals
9.Purification and Characterization of Transforming growth factor - beta1 from Human Platelets.
Eun Jo KO ; Jong Won LEE ; Sang Uk NHAM ; Eui Yul CHOI ; Gie Taek CHUN ; Se Won YIE ; Pyeung Hyeun KIM
Korean Journal of Immunology 1998;20(1):1-8
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
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Biological Assay
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Blood Platelets
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Cell Cycle
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Cell Differentiation
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Chromatography
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Chromatography, Gel
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Chromatography, High Pressure Liquid
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Chromatography, Liquid
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Electrophoresis
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Enzyme-Linked Immunosorbent Assay
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Humans*
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Immunoglobulin A
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Lung
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Mink
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Molecular Weight
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Silver Staining
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Spleen
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Transforming Growth Factors*
10.Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells.
Mi Hee PARK ; Yu Jin JUNG ; Pyeung Hyeun KIM
Immune Network 2012;12(1):27-32
BACKGROUND: Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. METHODS: In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. RESULTS: First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-beta1-induced germ line transcript alpha (GLTalpha) and IL-4-induced GLTgamma1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. CONCLUSION: Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.
B-Lymphocytes
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Cell Proliferation
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Germ Cells
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Immunoglobulin A
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Immunoglobulin Class Switching
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Immunoglobulin G
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Oligodeoxyribonucleotides
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Recombination, Genetic
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Toll-Like Receptors