OBJECTIVETo observe the in vivo antitumor activity of murine liver tumor vaccine expressing MIP-1alpha mediated by recombinant adenoviral vector.

METHODSThe infection efficacy was measured by GFP expression 48 hours after infection of Hepa1-6, and the number of cells was counted daily for 14 days. 5 x 10(6) modified Hepa1-6 cells were inoculated subcutaneously to C57BL/6 mice and the tumor-free animals were rechallenged by 2 x 10(6) wild-type Hepa1-6 cells or syngenic EL4 cells four weeks later. The tumor volume was measured twice a week.

RESULTSAdenoviral vectors could efficiently infect Hepa1-6 cells in vitro, and the in vitro growth rate of AdmMIP-1alpha modified Hepa1-6 cells was not affected; however the in vivo tumorigenicity was significantly decreased, compared with that of control vector modified Hepa1-6. Rechallenge of the tumor-free mice four weeks after administration of AdmMIP-1alpha with the parental Hepa1-6 cells resulted in significant inhibition of tumor growth, but there was no significant difference when rechallenged with EL4.

CONCLUSIONSThe liver cancer cells expressing mMIP-1alpha mediated by recombinant adenoviral vector decrease tumorigenicity and elicit specific immunological protection, and could be used as an effective liver tumor vaccine.

Adenoviridae ; genetics ; Animals ; Cancer Vaccines ; immunology ; Chemokine CCL3 ; Chemokine CCL4 ; Genetic Therapy ; Liver Neoplasms, Experimental ; therapy ; Macrophage Inflammatory Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Vaccines, Synthetic ; immunology

UNLABELLEDBACKGROWND: Macrophage inflammatory protein-1α (MIP-l α/CCL3) belongs to the C-C chemokine family (CCL3), which can be secreted by macrophages, other types of hematopoietic cells and bone marrow stromal cells. Higher levels of MIP-1α were found to be associated with several kinds of hematologic malignancies, including multiple myeloma (MM), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Moreover, MIP-1α has been reported to be an adverse prognostic factor for CLL. However, the impact of MIP-1α on acute myeloid leukemia (AML) has been poorly investigated.

OBJECTIVETo investigate the influence of MIP-1α on proliferction of AML cells.

METHODSUsing MLL-AF9 induced AML mouse model, the expression of MIP-1α was measured by real time quantitative RT-PCR. AML cell proliferation was examined by cell counting and colony forming assay (CFC). The influence of blocking the MIP-1α action on the growth and pathogenic ability of AML cells was explored by using the small molecule antagonist for interfering interaction of MIP-1α with its receptor CCR1.

RESULTSThe MIP-1α could promote the proliferation and colony formation of AML cells, the blocking MIP-1a could inhibit the growth of AML cells and delay onset of AML.

CONCLUSIONThe MIP-1a promotes the occurence and progression of AML, therefore blocking the MIP-1α signal pathway may be served as a strategy to inhibit the growth of AML cells, and MIP-1α can be a potential target for treatment of AML.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Chemokine CCL3 ; Chemokine CCL4 ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukemia, Myeloid, Acute ; Macrophage Inflammatory Proteins ; Mice ; Multiple Myeloma ; Receptors, CCR1

Animals ; Chemokine CCL20 ; Chemokine CXCL10 ; Chemokines, CC ; biosynthesis ; Chemokines, CXC ; biosynthesis ; Liver ; metabolism ; Liver Transplantation ; Macrophage Inflammatory Proteins ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Transplantation, Homologous

PURPOSE: The pathologic mechanisms of central nervous system(CNS) injuries in human meningitis are not yet completely understood. Recent studies indicate that the host inflammatory responses are as important in brain damage as the infecting organisms and toxins. There have been some reports on the relationship of nitric oxide(NO), macrophage inflammatory protein-1 alpha (MIP-1 alpha ), and lactoferrin in bacterial meningitis, but few reports in aseptic meningitis. Thus, we investigated the concentrations of NO, MIP-1 alpha and lactoferrin in cerebrospinal fluid(CSF) and serum of patients with aseptic meningitis and control subjects and evaluated their relationship with other parameters of meningitis. METHODS: CSF and blood were obtained from 25 subjects with aseptic meningitis and 15 control subjects. After centrifugation, supernatants were stored at -70degrees C and we assayed the concentrations of NO, MIP-1 alpha and lactoferrin with the ELISA method. There were no patients with neurologic sequelae after being recovered from aseptic meningitis. RESULTS: Concentrations of CSF and serum NO, MIP-1 alpha were not increased in aseptic meningitis subjects compared to control subjects. Concentration of CSF lactoferrin was significantly elevated in patients with aseptic meningitis and concentration of serum lactoferrin was significantly decreased in patients with aseptic meningitis compared with those in control subjects(P<0.05). CSF lactoferrin level was positively correlated with CSF WBC counts(rs=0.449, P=0.007), especially with neutrophil counts(rs=0.574, P<0.001) and CSF protein level(rs=0.508, P=0.002). CONCLUSION: Lactoferrin plays an important role in aseptic meningitis and may be released from neutrophils recruited from blood to the CSF through breakdown of blood-brain barrier. NO and MIP-1 alpha may not be important factors in the pathogenesis of aseptic meningitis without neurologic sequelae.
Blood-Brain Barrier ; Brain ; Centrifugation ; Enzyme-Linked Immunosorbent Assay ; Humans ; Lactoferrin* ; Macrophage Inflammatory Proteins* ; Macrophages* ; Meningitis ; Meningitis, Aseptic* ; Meningitis, Bacterial ; Neutrophils ; Nitric Oxide*

BACKGROUND: The induction of production and production inhibition of alpha1-antichymotrypsin (ACT), IL-1 alpha, IL-1 alpha receptor and macrophage inflammatory protein-1 (MIP-1) receptor in A beta1-42 (A beta)-stimulated U373MG cell, the human astrocytoma cell line, have never been reported. METHODS: U373MG cells (1 x 10(6) cells in RPMI-1640 media) were incubated for overnight after administration of a single dose of 20 micro M of A beta or 0.5 ng/ml of TNF alpha or both. Actinomycin D (2.5 micro M) or cycloheximide (2.5 micro M) was also added to the cell suspension. Messenger RNA expression of ACT, IL-1 alpha, IL-1 alpha receptor and MIP-1 receptor was measured by RT-PCR. Western blot was done and nitrocellulose paper was stained with anti-ACT and anti-GFAP antibody. NF kappa B activation after treatment of A beta in U373MG cells was detected by electrophoretic mobility-shift assay. RESULTS: A beta and TNF alpha both increased production of ACT in a dose-dependent manner. TNF alpha enhanced A beta-induced mRNA had increases of ACT, IL-1 alpha, IL-1 alpha receptor and MIP-1 alpha receptor. Activated NF kappa B was demonstrated in the A beta, TNF alpha-stimulated U373MG cells. Actinomycin suppresses mRNA level of ACT and IL-1 alpha receptor but cycloheximide inhibits the expression of ACT, IL-1 alpha and MIP-1 alpha receptor. CONCLUSIONS: TNF alpha increases synthesis of ACT, IL-1 alpha, IL-1 alpha receptor and MIP-1 alpha receptor in A beta-stimulated astrocyte, which, as a result, may contribute to the neuroinflammation of Alzheimer's disease.
Alzheimer Disease ; Amyloid beta-Peptides* ; Astrocytes ; Astrocytoma* ; Blotting, Western ; Cell Line* ; Collodion ; Cycloheximide ; Dactinomycin ; Humans* ; Interleukin-1 ; Interleukin-1alpha ; Macrophage Inflammatory Proteins ; NF-kappa B ; RNA, Messenger

Nystatin, a polyene antifungal antibiotic, is a cholesterol sequestering agent. The antifungal agent alters composition of the plasma membrane of eukaryotic cells, whereas its effects on cells are poorly investigated. In the current study, we investigated the question of whether nystatin was able to induce expression of macrophage inflammatory protein-1 (MIP-1). THP-1 cells rarely express MIP-1alpha and MIP-1beta, however, upon exposure to nystatin, significantly elevated expression of MIP-1alpha and MIP-1beta was observed in a dose-dependent fashion at the messenger and protein levels. Cellular factors activated by nystatin as well as involved in nystatin-induced expression of MIP-1 proteins were identified in order to understand the molecular mechanisms of action of the anti-fungal agent. Treatment with nystatin resulted in enhanced phosphorylation of Akt, ERK, p38 MAPK, and JNK. Abrogation or significant attenuation of nystatin-induced expression of MIP-1alpha and MIP-1beta was observed by treatment with Akt inhibitor IV, LY294002, and SP6001250. Inhibition of ERK or p38MAPK using U0126 and SB202190 did not lead to attenuation of MIP-1 expression. In addition, inhibitors of protein kinase C, such as GF109203X and Ro-318220, also attenuated expression of MIP-1. These results indicate that nystatin is able to activate multiple cellular kinases and, among them, Akt and JNK play primary roles in nystatin-induced expression of MIP-1 proteins.
Cell Membrane ; Chemokine CCL3 ; Chemokine CCL4 ; Cholesterol ; Eukaryotic Cells ; Macrophage Inflammatory Proteins* ; Macrophages* ; Nystatin* ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Phosphotransferases ; Protein Kinase C

OBJECTIVETo study the effect of diamide on the expression of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in cultured human umbilical vein endothelial cells.

METHODSAfter exposure of the endothelial cells (ECs) to different concentrations of diamide for 4 hours, the MIP-1 alpha mRNA in the cells was detected by nuclease S1 protection assay and the MIP-1 alpha protein in those cells was determined by cell enzyme-linked immunosorbent assay. The chemotactic activity of MIP-1 alpha in the conditioned medium of ECs treated with diamide for peripheral blood monocytes was tested by microfilter method using modified Boyden chambers.

RESULTSIncubation of ECs with 5 micro mol/L diamide resulted in a 2.4-fold increase in the level of MIP-1 alpha mRNA expression as compared with the control group (t = 8.70, P < 0.05). Exposure of ECs to 1 micro mol/L, 5 micro mol/L and 10 micro mol/L diamide resulted in a 0.9-fold, 1.2-fold, and 0.7-fold increase in the level of MIP-1 alpha protein expression respectively, as compared with the control group (F = 35.65, P < 0.05). Chemotactic assay showed that the migration distance of monocytes towards the conditioned medium (CM) of ECs treated with 5 micromol/L diamide was 99.50 microm +/- 4.31 microm, which was significantly more than the 66.47 microm +/- 3.25 microm towards the conditioned medium of ECs in the non-diamide group, the chemokinetic group (67.03 microm +/- 6.83 microm) and the random migration group (65.40 microm +/- 3.36 microm) (F = 404.31, P < 0.05). The results revealed that there might be chemotactic substances in the conditioned medium of 5 micro mol/L diamide treated ECs. The migration distance of monocytes towards the conditioned medium of the ECs exposed to 5 micromol/L diamide was significantly reduced to 82.80 microm +/- 6.88 microm after the addition of goat anti-human MIP-1 alpha antibody (F = 192.25, P < 0.05), which indicates the chemotactic activity of MIP-1 alpha in the conditioned medium of the ECs in the diamide group.

CONCLUSIONSDiamide, a lipid peroxidation inducer, could stimulate ECs to produce high levels of MIP-1 alpha with chemotactic activity, and may play an important role in atherogenesis through attraction of peripheral blood monocytes into arterial intima.

Arteriosclerosis ; pathology ; Cells, Cultured ; Chemokine CCL4 ; Diamide ; pharmacology ; Endothelium, Vascular ; drug effects ; metabolism ; Gene Expression ; drug effects ; Humans ; Macrophage Inflammatory Proteins ; metabolism ; RNA, Messenger ; drug effects ; metabolism ; Radiation-Sensitizing Agents ; pharmacology

To explain biological function of protein CCL15 in HCC cell lines. The different expression level of CCL15 among HCC cell lines was validated by RT-PCR and Western blot. The expression recombinant plasmid of siRNA-CCL15 was constructed successfully and transfected into high metastasis cell lines HCCML3 to observe the alteration of biological function of HCCML3. The overexpression of CCL15 in high metastasis HCC cell lines was confirmed by validation tests. After transfected with siRNA-CCL15, the average amounts of invaded cells in cell invasion assay were 657.9 (HCCML3) and 148.4(HCCML3-siCCL15) (t=19.34, P less than 0.05). And in the scratch assay, the migrating distance were (0.35+/-0.02) mm (HCCML3) and (0.82+/-0.03)mm (HCCML3-siCCL15) (t=15.67, P less than 0.05). The expression of MMP-9 in HCCML3 was higher than HCCML3-siCCL15 through Western blot. Some biological properties (migration, invasion, MMP-9) of HCCML3 transfected with siRNA-CCL15 were decreased. The results suggest CCL15 might play an important role in HCC cell invasion and metastasis through two paths of MMPs regulation and invasion potential strengthening.
Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Chemokines, CC ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Macrophage Inflammatory Proteins ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Transfection

OBJECTIVETo study the effect of macrophage inflammatory protein-1alpha(MIP-1alpha) and its mRNA on airway inflammation of mouse with induced asthma.

METHODSSeventy male BALB/C mice were randomly divided into the control group and asthma group (including 7 subgroups, 10 mice each). The control group included group A(24) (the lavaging subgroup was sacrificed 24 h after the last challenge) and group A(0) (the non-lavaging subgroup was sacrificed from 18 h to 24 h after the last challenge); asthma group included group B(3) (the lavaging subgroup was sacrificed 3 h after the last challenge), group B(8) (the lavaging subgroup was sacrificed 8 h after the last challenge), group B(24) (the lavaging subgroup was sacrificed 24 h after the last challenge), group B(36) (the lavaging subgroup was sacrificed 36 h after the last challenge) and group B(0) (the non-lavaging subgroup was sacrificed from 18 h to 24 h after the last challenge). In the experiment, the mice model of asthma was established by the ovalbumin (OVA) challenge methods. Eosinophils (EOS) numbers and differentiated cell numbers in bronchoalveolar lavage fluid (BALF) were counted; the concentrations of MIP-1alpha in serum and BALF were measured by sandwich enzyme-linked immunosorbent assay (sandwich ELISA); the protein expressions of MIP-1alpha were detected by immunohistochemical techniques; the mRNA expressions of MIP-1alpha were determined by in situ hybridization technique.

RESULTS(1) The concentrations of MIP-1alpha in BALF and serum of group B(3) [(30.2 +/- 4.2) pg/ml, (30.8 +/- 4.6) pg/ml], group B(8) [(35.3 +/- 4.9) pg/ml, (34.9 +/- 5.1) pg/ml], group B(24) [(42.9 +/- 5.8) pg/ml, (41.7 +/- 6.3) pg/ml] and group B(36) [(37.8 +/- 4.7) pg/ml, (35.7 +/- 4.9) pg/ml] were significantly higher than those of group A(24) [(20.9 +/- 3.8) pg/ml, (22.4 +/- 4.3) pg/ml] (P < 0.01); the concentrations of MIP-1alpha in BALF and serum went up at 3 h, reached peak at 24 h, and had descended at 36 h. (2) Immunohistochemistry showed that the protein expressions of MIP-1alpha around the bronchus of group B(0) [(26.4 +/- 6.2)%] were significantly elevated as compared to those of group A(0) [(10.3 +/- 2.5)%] (P < 0.01), the epithelial cell was the chief expression cell. (3) In situ hybridization showed that the mRNA expressions of MIP-1alpha around the bronchus of group B(0) [(23.9 +/- 4.2)%] were significantly increased when compared to those of group A(0) [(8.7 +/- 1.8)%] (P < 0.01), the epithelial cell was the chief expression cell. (4) There was a significant correlation between the concentrations of MIP-1alpha and the numbers of EOS in BALF and between the concentrations of MIP-1alpha and the percentage of EOS numbers in the total cell numbers (EOS%) in BALF.

CONCLUSIONSMIP-1alpha protein and MIP-1alpha mRNA were found strongly expressed in mouse asthma model, the epithelial cell was the chief expression cell; the kinetic characteristic of MIP-1alpha showed that its level increased at 3 h, reached peak at 24 h and declined at 36 h; MIP-1alpha and EOS gathering had a significant correlation.

Animals ; Asthma ; blood ; genetics ; Bronchoalveolar Lavage Fluid ; chemistry ; Chemokine CCL3 ; Chemokine CCL4 ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; In Situ Hybridization ; Macrophage Inflammatory Proteins ; blood ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; genetics ; metabolism

Cells, Cultured ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; drug effects ; Endothelial Cells ; cytology ; metabolism ; Homocysteine ; pharmacology ; Humans ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Monocytes ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Umbilical Veins ; cytology