BACKGROUND: We investigated whether non-steroidal anti-inflammatory drugs(NSAIDs) could influence the expression of a few inflammatory mediator-related genes in amyloid-beta1-42(Abeta42)-activated microglia. METHODS: BV-2 cells, a murine microglial cell line, were pretreated with a single dose of 20microM of aggregated Abeta42 for 18 hours followed by incubation with ibuprofen(100microM), indomethacin(150microM) or ketorolac(10nM) for 24 hours. Expression of mRNAs for CCL7(beta-chemokine), CXCL2(alpha-chemokine), CCR7(beta-chemokine receptor), interleukin(IL)-1alpha, matrix metalloproteinase(MMP)-3, beta-secretase(BACE1) and cyclooxygenase(COX)-2 gene were measured with quantitative realtime reverse transcriptase(RT)-PCR. RESULTS: Abeta42 increased expression of mRNAs for CCL7, CXCL2, CCR7, IL-1alpha, MMP-3, BACE1 and COX-2 genes. Administration of each NSAIDs effectively lowered the expression of these genes in Abeta42-activated microglia. CONCLUSION: NSAIDs inhibit increased expression of a few cytokines, chemokine receptor and inflammatory mediatorrelated protease genes in Abeta42-activated microglia. These data demonstrate a possible mechanism how NSAIDS may decrease the risk and delay the onset of chronic neuroinflammatory process in AD.
Alzheimer Disease ; Amyloid Precursor Protein Secretases ; Animals ; Anti-Inflammatory Agents, Non-Steroidal ; Cell Line ; Chemokine CCL7 ; Cytokines ; Gene Expression* ; Mice* ; Microglia ; RNA, Messenger

OBJECTIVE: To investigate the mRNA level of IL-12, ICAM-1 and MCP-3 in human nasal epithelial cells. METHOD: Firstly, human primary nasal epithelial cells were cultured, and then 4 pairs of primers were designed for detecting mRNA level of IL-12, ICAM-1 and MCP-3. The 938 bp PCR products of GAPDH were used as internal standards. The mRNA expression levels of IL-12, ICAM-1 and MCP-3 in primary nasal epithelial cells was measured with semi-quantitative reverse transcription-polymerase chain reaction. RESULT: The round or irregular primary nasal epithelial cells were observed sticking to the bottom of cell culture plates under 400 times optical microscope. The expressions of IL-12 p35, ICAM-1 and MCP-3 mRNA were found in primary nasal epithelial cells while IL-12 p40 subunit was not detected. CONCLUSION IL-12 p35, ICAM-1 and MCP-3 mRNAs are expressed in primary nasal epithelial cells, whereas effective IL-12 with integrity is not present in nasal epithelial cells.
Cells, Cultured ; Chemokine CCL7 ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-12 Subunit p35 ; metabolism ; Nasal Mucosa ; cytology ; metabolism ; RNA, Messenger ; genetics

OBJECTIVE: To investigate monocyte chemotactic protein-3 (MCP-3) mRNA and protein expression and distribution in laryngeal squamous cell carcinoma (LSCC) tissue, so as to study its function in the tumor development. METHOD: Immunohistochemical staining and RT-PCR, and western blot were performed to detect the LSCC tissue and the normal tissue adjacent tumor. RESULT: The expression of MCP-3 mRNA and protein were found in the LSCC tissue and the normal tissue adjacent tumor. The expression of MCP-3 mRNA and protein in LSCC tissue is much higher than that in normal tissue adjacent tumor (P<0.05). There is a significant difference between I-II stage III-IV stage (P<0.05). Immunohistochemical staining: positive staining of MCP-3 was yellow and mainly detected in the tumor cell cytoplasm and also in some normal glandular epitheliums. CONCLUSION MCP-3 might be involved in the mechanism of the tumor development, its function of chemotaxis and activation of the tumor-associated macrophages would be highlighted, so that the inflammatory microenvironment might play an important role in the development of tumor.
Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Chemokine CCL7 ; metabolism ; Female ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; genetics

The idiotypic determinant of surface immunoglobulin of B-cell lymphoma, as a tumor-specific antigen, has proved to be able to induce immune responses. To analyze whether an idiotypic vaccine fused with cytokine can elicit more effectively protective antitumor immunity, an eukaryotic expression plasmid was constructed, which encoded the fusion gene of single-chain variable fragment as a tumor specific antigen against B-cell lymphoma with monocyte chemotactic protein-3 (MCP3) as immunogen to elicit T-cell-dependent protective antitumor immunity, and EGFP (Enhanced Green Fluorescent Protein) gene as a marker to trace the survival, growth, differentiation and expression of the former exogenetic genes. The cDNAs for immunoglobulin (Ig) VH and IgVL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) connected with a (Gly(4)Ser)(3) linker by recombinant PCR method. Then, the fragments of scFv and MCP3 were ligated with a NDAQAPKS spacer by the same method. The results showed that the fusion genes of scFv and MCP3-scFv were inserted into an eukaryotic expression vector pTARGET, and EGFP was cloned into the downstream of scFv and MCP3-scFv respectively. Finally the constructed plasmids were confirmed by sequencing and restriction analysis. In conclusion, a tumor-derived idiotypic DNA vaccine, encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP3) to elicit a T-cell dependent, antitumor immunity, and the EGFP gene was inserted correctly. The DNA vaccine could be used for further study of DNA vaccine against B cell lymphoma in vivo.
Animals ; Cancer Vaccines ; genetics ; immunology ; Cell Line, Tumor ; Chemokine CCL7 ; immunology ; Genetic Vectors ; Immunoglobulin Variable Region ; immunology ; Lymphoma, B-Cell ; genetics ; immunology ; prevention & control ; Mice ; Mice, Inbred BALB C ; Plasmids ; Single-Chain Antibodies ; immunology ; Vaccines, DNA ; genetics ; immunology

OBJECTIVETo observe the effects of epimedium herb (EH), a Chinese herb for replenishing Shen, on regulated on activation, normal T call expressed and secreted (RANTES) and monocyte chemotactic protein-3 (MCP-3) expression in lung tissue of asthmatic rats, for further exploring the action mechanism of EH in treating asthma.

METHODSBrown Norway rats were randomly divided into six groups: the normal control group (A), the allergic asthma model group (B), the group of model rat treated with dexamethasone (C), and the three groups of model rat treated with low (0.125 g/mL), medium (0.5 g/mL), high (2.0 g/mL) dose of EH (D, E and F). RANTES and MCP-3 mRNA expressions in lung tissue were tested with Real-time PCR and the serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin-4 (IL-4), and interleukin-5 (IL-5) were measured by enzyme linked immunosorbent assay (ELISA).

RESULTSAs compared with the model group (B), RANTES, MCP-3 expression and TNF-alpha were lower in the 4 treated groups (C, D, E and F, P < 0.05 or P < 0.01); IL-4 was lower in Group C, E and F (P = 0.007, P = 0.047, P = 0.033), while that in Group D was higher than that in Group C (P = 0.012). As for level of IL-5, lowering was shown only in Group C and F (P = 0.003, P = 0.005).

CONCLUSIONApplying EH in the attack stage of asthma can alleviate the airway inflammation by down-regulating the expression of RANTES and MCP-3 in lung tissue.

Animals ; Asthma ; blood ; physiopathology ; prevention & control ; Chemokine CCL5 ; genetics ; Chemokine CCL7 ; genetics ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Epimedium ; chemistry ; Gene Expression ; drug effects ; Interleukin-4 ; blood ; Interleukin-5 ; blood ; Lung ; drug effects ; metabolism ; pathology ; Male ; Phytotherapy ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Inbred BN ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo investigate the effect of monocyte chemotactic protein-3 (MCP-3) on the expressions of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tissue factor (TF, and tissue factor pathway inhibitor (TFPI) and cell apoptosis in human umbilical vein endothelial cells (HUVECs).

METHODSCultured HUVECs were treated with MCP-3 at the optimal concentration determined previously 1 h after treatments with or without MCP-3 antibody (20 ng/ml), PI3K inhibitor, or LY-294002 (5 mmol/ml). The expressions of ICAM-1, VCAM-1, TF and TFPI were analyzed using RT-PCR and Western blot after the treatments. MCP-3 mRNA and protein expressions were detected in HUVECs exposed to 50 µg/ml ox-LDL for 24 h. The cell apoptosis and caspase-3 protein production in HUVECs treated with MCP-3 or with MCP-3 plus CCR2 antagonist for 24 h and 48 h were evaluated by flow cytometry and Western blotting.

RESULTSAt the optimal concentration of 0.3 ng/ml, MCP-3 treatment for 24 h caused significantly increased ICAM-1, VCAM-1, and TF expressions with lowered expression of TFPI in HUVECs (P<0.05), and such effects were significantly inhibited by the application of MCP-3 antibody, PI3K inhibitor, or LY-294002 (P<0.05). Ox-LDL exposure significantly increased the expression of MCP-3 in HUVECs (P<0.05). HUVECs showed a significantly increased apoptosis rate after treatment with MCP-3 or with MCP-3 plus CCR2 antagonist (P<0.05), and the apoptosis rate increased significantly as the treatment time prolonged (P<0.05); caspase-3 protein expression in the cells showed a similar pattern of alterations following the treatments.

CONCLUSIONox-LDL can induce MCP-3 expression in HUVECs. MCP-3 induces apoptosis of HUVECs and significantly affects the cellular function partially through the PI3K signaling pathway.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Adhesion ; Cells, Cultured ; Chemokine CCL7 ; pharmacology ; Chromones ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipoproteins ; metabolism ; Lipoproteins, LDL ; pharmacology ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Receptors, CCR2 ; antagonists & inhibitors ; Signal Transduction ; Thromboplastin ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism

The aim was to construct a prokaryotic expression plasmid encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP-3). The cDNAs of immunoglobulin (Ig) VH and Ig VL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) by recombinant PCR method. The cDNAs of Ig VH and Ig VL were connected by a (Gly4Ser)3 linker. Then, the fragments of scFv and MCP-3 were connected with a NDAQAPKS spacer, using recombinant PCR method again. The results indicated that the fusion gene of scFv-MCP-3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases examination. Finally, the fusion protein was expressed in E coli DH5alpha under induction by arabinose. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria. In conclusion, a prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP-3 and expressing idiotype protein vaccination against B cell lymphoma, was constructed correctly.
Amino Acid Sequence ; Animals ; Cancer Vaccines ; biosynthesis ; genetics ; immunology ; Chemokine CCL7 ; biosynthesis ; genetics ; immunology ; Genetic Vectors ; Humans ; Immunoglobulin Idiotypes ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Lymphoma, B-Cell ; immunology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, DNA ; biosynthesis ; genetics ; immunology

The purpose of this study was to evaluate whether the DNA vaccine containing idiotypic gene fragment of human B-cell lymphoma cell line Namalwa could elicit the specific anti-idiotypic immune response in vivo. The candidate gene fragment of the lymphoma cell, variable region of heavy chain (VH) of the membranous immunoglobulin, was amplified using Ig superfamily primers by means of RT-PCR. Also, the intact cDNA of murine monocyte chemoattractant protein (MCP-3) was cloned and used as the adjuvant molecular. The two gene fragments of VH and MCP-3 were fused together by 8aa linker peptide with recombinant PCR. Subsequently, the fusion gene fragment was cloned into eukaryonic expression vector pcDNA3.1 to construct DNA vaccine plasmid. Prior to the immunization, the transient transfection coupled with RT-PCR was performed to prove that the recombinant plasmid could express in eukaryonic cells in right way. Then two groups of mice were immunized by intramuscular injection with DNA vaccine and mock plasmid pcDNA3.1 respectively. Three times of injection were performed with 100 micro g plasmid respectively at the beginning of the experiment and 2, 4 weeks after the first injection for all the groups. FACS analysis was chosen to detect the antibodies recognizing lymphoma cells at different time following vaccination. The results demonstrated that specific anti-idiotypic antibody could be detected in the group of DNA vaccine immunized mice as early as eight weeks after the first immunization. Further test demonstrated that the anti-idiotypic antibody could maintain for at least twenty weeks with high titer. Anti-idiotypic antibodies were elicited in three of five mice of the DNA vaccine immunized group. The Abs of DNA vaccine immunized mice could only recognize Namalwa cell line instead of another unrelated human cell line A549. There is no cellular response detected in the DNA vaccine immunized mice. It is concluded that the DNA vaccine containing fused MCP3-VH sequence could elicit specific anti-idiotypic antibody against B-cell lymphoma in vivo and could be used in further study of DNA vaccine against B-cell lymphoma. The results would provide the basis for further studies and optimization of this therapeutic strategy on patients with B-lymphoproliferative disease.
Animals ; Antibodies, Anti-Idiotypic ; blood ; COS Cells ; Cancer Vaccines ; immunology ; Chemokine CCL7 ; Cytokines ; Humans ; Immunization ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lymphoma, B-Cell ; immunology ; Mice ; Mice, Inbred BALB C ; Monocyte Chemoattractant Proteins ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, DNA ; immunology