1.Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59.
Jeong Won HONG ; Woon Dong CHO ; Kwon Pyo HONG ; So Seul KIM ; Seung Myoung SON ; Seok Joong YUN ; Ho Chang LEE ; Sang Soon YOON ; Hyung Geun SONG
Immune Network 2012;12(1):33-39
BACKGROUND: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). METHODS: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reverse-transcriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. RESULTS: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. CONCLUSION: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.
Animals
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Antibodies, Monoclonal
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Base Sequence
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Cell Line
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Centers for Disease Control and Prevention (U.S.)
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Clone Cells
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Complement System Proteins
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Flow Cytometry
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Humans
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Hybridomas
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Light
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Mice
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Polymerase Chain Reaction
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Prostatic Neoplasms
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Proteins
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RNA, Messenger
2.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
3.The Analysis of Vitamin C Concentration in Organs of Gulo-/- Mice Upon Vitamin C Withdrawal.
Hyemin KIM ; Seyeon BAE ; Yeonsil YU ; Yejin KIM ; Hang Rae KIM ; Young il HWANG ; Jae Seung KANG ; Wang Jae LEE
Immune Network 2012;12(1):18-26
BACKGROUND: Vitamin C is an essential nutrient for maintaining human life. Vitamin C insufficiency in the plasma is closely related with the development of scurvy. However, in vivo kinetics of vitamin C regarding its storage and consumption is still largely unknown. METHODS: We used Gulo-/- mice, which cannot synthesize vitamin C like human. Vitamin C level in plasma and organs from Gulo-/- mice was examined, and it compared with the level of wild-type mice during 5 weeks. RESULTS: The significant weight loss of Gulo-/- mice was shown at 3 weeks after vitamin C withdrawal. However, there was no differences between wild-type and vitamin C-supplemented Gulo-/- mice (3.3 g/L in drinking water). The concentration of vitamin C in plasma and organs was significantly decreased at 1 week after vitamin C withdrawal. Vitamin C is preferentially deposited in adrenal gland, lymph node, lung, and brain. There were no significant changes in the numbers and CD4/CD8 ratio of splenocytes in Gulo-/- mice with vitamin C withdrawal for 4 weeks. And the architecture of spleen in Gulo-/- mice was disrupted at 5 weeks after vitamin C withdrawal. CONCLUSION: The vitamin C level of Gulo-/- mice was considerably decreased from 1 week after vitamin C withdrawal. Vitamin C is preferentially stored in some organs such as brain, adrenal gland and lung.
Adrenal Glands
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Animals
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Ascorbic Acid
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Brain
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Drinking
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Humans
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Kinetics
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Lung
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Lymph Nodes
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Mice
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Plasma
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Scurvy
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Spleen
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Vitamins
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Weight Loss
4.Baculovirus-based Vaccine Displaying Respiratory Syncytial Virus Glycoprotein Induces Protective Immunity against RSV Infection without Vaccine-Enhanced Disease.
Immune Network 2012;12(1):8-17
BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract diseases in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV yet. The attachment glycoprotein (G) of RSV is a potentially important target for protective antiviral immune responses. Recombinant baculovirus has been recently emerged as a new vaccine vector, since it has intrinsic immunostimulatory properties and good bio-safety profile. METHODS: We have constructed a recombinant baculovirus-based RSV vaccine, Bac-RSV/G, displaying G glycoprotein, and evaluated immunogenicity and protective efficacy by intranasal immunization of BALB/c mice with Bac-RSV/G. RESULTS: Bac-RSV/G efficiently provides protective immunity against RSV challenge. Strong serum IgG and mucosal IgA responses were induced by intranasal immunization with Bac-RSV/G. In addition to humoral immunity, G-specific Th17- as well as Th1-type T-cell responses were detected in the lungs of Bac-RSV/G-immune mice upon RSV challenge. Neither lung eosinophilia nor vaccine-induced weight loss was observed upon Bac-RSV/G immunization and subsequent RSV infection. CONCLUSION: Our data demonstrate that intranasal administration of baculovirus-based Bac-RSV/G vaccine is efficient for the induction of protection against RSV and represents a promising prophylactic vaccination regimen.
Administration, Intranasal
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Animals
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Baculoviridae
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Eosinophilia
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Glycoproteins
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Immunity, Humoral
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Immunization
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Immunoglobulin A
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Immunoglobulin G
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Lung
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Mice
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Respiratory Syncytial Viruses
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Respiratory Tract Diseases
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T-Lymphocytes
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Vaccination
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Weight Loss
5.Molecular Mechanisms Governing IL-24 Gene Expression.
Immune Network 2012;12(1):1-7
Interleukin-24 (IL-24) belongs to the IL-10 family of cytokines and is well known for its tumor suppressor activity. This cytokine is released by both immune and nonimmune cells and acts on non-hematopoietic tissues such as skin, lung and reproductive tissues. Apart from its ubiquitous tumor suppressor function, IL-24 is also known to be involved in the immunopathology of autoimmune diseases like psoriasis and rheumatoid arthritis. Although the cellular sources and functions of IL-24 are being increasingly investigated, the molecular mechanisms of IL-24 gene expression at the levels of signal transduction, epigenetics and transcription factor binding are still unclear. Understanding the specific molecular events that regulate the production of IL-24 will help to answer the remaining questions that are important for the design of new strategies of immune intervention involving IL-24. Herein, we briefly review the signaling pathways and transcription factors that facilitate, induce, or repress production of this cytokine along with the cellular sources and functions of IL-24.
Arthritis, Rheumatoid
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Autoimmune Diseases
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Chromatin Assembly and Disassembly
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Cytokines
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Epigenomics
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Gene Expression
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Humans
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Interleukin-10
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Interleukins
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Lung
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Psoriasis
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Signal Transduction
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Skin
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Transcription Factors
6.IGF-I Exerts an Anti-inflammatory Effect on Skeletal Muscle Cells through Down-regulation of TLR4 Signaling.
Immune Network 2011;11(4):223-226
Although exercise-induced growth factors such as Insulin-like growth factor-I (IGF-I) are known to affect various aspects of physiology in skeletal muscle cells, the molecular mechanism by which IGF-I modulates anti-inflammatory effects in these cells is presently unknown. Here, we showed that IGF-I stimulation suppresses the expression of toll-like receptor 4 (TLR4), a key innate immune receptor. A pharmacological inhibitor study further showed that PI3K/Akt signaling pathway is required for IGF-I-mediated negative regulation of TLR4 expression. Furthermore, IGF-I treatment reduced the expression of various NF-kappaB-target genes such as TNF-alpha and IL-6. Taken together, these findings indicate that the anti-inflammatory effect of exercise may be due, at least in part, to IGF-I-induced suppression of TLR4 and subsequent downregulation of the TLR4-dependent inflammatory signaling pathway.
Cytokines
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Down-Regulation
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Insulin-Like Growth Factor I
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Intercellular Signaling Peptides and Proteins
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Interleukin-6
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Muscle, Skeletal
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Toll-Like Receptor 4
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Tumor Necrosis Factor-alpha
7.Recombinant TAT-CD137 Ligand Cytoplasmic Domain Fusion Protein Induces the Production of IL-6 and TNF-alpha in Peritoneal Macrophages.
Jung Dae KIM ; Eun Ah LEE ; Nguyen N QUANG ; Hong Rae CHO ; Byungsuk KWON
Immune Network 2011;11(4):216-222
BACKGROUND: The ligand for CD137 (CD137L; also called 4-1BBL) is mainly expressed on activated APCs such as dendritic cells, B cells and macrophages. Even though CD137L functions as a trigger of the CD137 signaling pathway for T cell activation and expansion, engagement of CD137L can deliver a signal leading to the production of proinflammatory cytokines in macrophages. METHODS: We generated cell-permeable TAT-CD137L cytoplasmic domain fusion protein (TAT-CD137Lct) and examined its ability to initiate the CD137L reverse signaling pathway. RESULTS: Treatment of TAT-CD137Lct induced the production of high levels of IL-6 and TNF-alpha mRNAs and proteins in peritoneal macrophages. TAT-CD137Lct increased phosphorylation of Erk, p38 MAPK and Jnk, and activated transcription factors C/EBP and CREB. However, TAT-CD137Lct did not visibly affect the degradation of the inhibitor of NF-kB (IkBalpha). We further demonstrated that JNK activation was required for TAT-CD137Lct-induced production of TNF-alpha, while activation of Erk and p38 MAPK were involved in IL-6 and TNF-alpha production. CONCLUSION: Our results suggest that TAT-CD137Lct is an effective activator for the CD137L reverse signaling pathway.
4-1BB Ligand
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B-Lymphocytes
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Cytokines
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Cytoplasm
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Dendritic Cells
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Interleukin-6
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Macrophages
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Macrophages, Peritoneal
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NF-kappa B
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p38 Mitogen-Activated Protein Kinases
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Phosphorylation
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Proteins
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RNA, Messenger
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Transcription Factors
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Tumor Necrosis Factor-alpha
8.The Anti-tumor Activity of Vitamin C via the Increase of Fas (CD95) and MHC I Expression on Human Stomach Cancer Cell Line, SNU1.
Yeonsil YU ; Seyeon BAE ; Hyemin KIM ; Yejin KIM ; Nag Bum CHU ; Nag Kyun CHU ; Jae Seung KANG ; Wang Jae LEE
Immune Network 2011;11(4):210-215
BACKGROUND: It is already known that high concentration of vitamin C induces apoptosis on tumor cells. However, there is no report regarding the function of vitamin C on the modulation of immune susceptibility of cancer. Therefore, we investigated whether vitamin C can modulate immune susceptibility of tumor cells, especially on the induction of Fas-mediated apoptosis. METHODS: First, the optimal concentration of vitamin C, which cannot induce damages on tumor cells for 36 hrs. We found that 2 mM of vitamin C did not show harmful effect. In addition, the optimal concentration of agonistic anti-Fas Abs for 18 hrs was examined. RESULTS: As a result, 400 ng/ml of agonistic anti-Fas Abs did not induce apoptosis on tumor cells. Next, we tried to find the effect of 2 mM of vitamin C on the modulation of the susceptibility to agonistic anti-Fas Abs. When tumor cells were cultured with 400 ng/ml of agonistic anti-Fas Abs for 18 hrs, after pre-treatment with 2 mM of vitamin C for 24 hrs, viability of cells was decreased. Interestingly, we found that the expression of Fas (CD95) and MHC class I was increased by the treatment of vitamin C. CONCLUSION: Taken together, vitamin C increases the susceptibility of tumor cells to anti-Fas Abs and the expression of Fas (CD95) and MHC class I on tumor cells.
Apoptosis
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Ascorbic Acid
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Cell Line
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Humans
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Stomach
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Stomach Neoplasms
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Vitamins
9.Identification of CCL1 as a Gene Differentially Expressed in CD4+ T Cells Expressing TIM-3.
Ka Jung JUN ; Mi Jin LEE ; Dong Chul SHIN ; Min Yeong WOO ; Kyongmin KIM ; Sun PARK
Immune Network 2011;11(4):203-209
BACKGROUND: T cell immunoglobulin mucin containing molecule (TIM)-3 is expressed in differentiated Th1 cells and is involved in the suppression of the cytokine production by these cells. However, the regulation of the expression of other T cell genes by TIM-3 is unclear. Herein, we attempted to identify differentially expressed genes in cells abundantly expressing TIM-3 compared to cells with low expression of TIM-3. METHODS: TIM-3 overexpressing cell clones were established by transfection of Jurkat T cells with TIM-3 expression vector. For screening of differentially expressed genes, gene fishing technology based on reverse transcription-polymerase chain reaction (RT-PCR) using an annealing control primer system was used. The selected candidate genes were validated by semi quantitative and real-time RT-PCR. RESULTS: The transcription of TIMP-1, IFITM1, PAR3 and CCL1 was different between TIM-3 overexpressing cells and control cells. However, only CCL1 transcription was significantly different in cells transiently transfected with TIM3 expression vector compared with control cells. CCL1 transcription was increased in primary human CD4+ T cells abundantly expressing TIM-3 but not in cells with low expression of TIM-3. CONCLUSION: CCL1 was identified as a differentially transcribed gene in TIM-3-expressing CD4+ T cells.
Clone Cells
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Gene Expression
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Genes, vif
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Humans
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Immunoglobulins
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Mass Screening
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Mucins
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T-Lymphocytes
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Th1 Cells
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Tissue Inhibitor of Metalloproteinase-1
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Transfection
10.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