OBJECTIVETo investigate the differential expression of isocitrate lyase in Penicillium marneffei phagocytized by nonstimulated and stimulated murine macrophages, and explore the role of glyoxylate pathway in pathogenesis of Penicilliosis marneffei.

METHODSPenicillium marneffei conidia and Raw264.7 cells were incubated in 16 cultures, which were divided to 4 groups for treatment with N-monomethyl-L-arginine (LNMMA, CI group), murine interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS) (T group), IFN-gamma plus LPS and LNMMA (TI group), or the same volume of culture medium (C group). The transcriptional levels of isocitrate lyase were detected using real-time RT-PCR, and its expression levels detected biochemically.

RESULTSThe transcriptional levels of isocitrate lyase in C, CI, T, TI groups were 1.00, 1.42, 33.09, and 74.88 (P<0.05), while the expression levels were 0.06, 0.07, 0.18, and 0.93, respectively (P<0.05). The content of nitric oxide in T group was significantly higher than that in the other groups (P<0.01), but the CFU of T group was the lowest (P<0.01).

CONCLUSIONReactive nitrogen intermediates induced by stimulated murine macrophages restrain the expression of isocitrate lyase of Penicillium marneffei and development of Penicillium marneffei, in which process the glyoxylate pathway may play an important role.

Animals ; Cell Line ; Fungal Proteins ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Enzymologic ; drug effects ; Gene Expression Regulation, Fungal ; drug effects ; Host-Pathogen Interactions ; Interferon-gamma ; pharmacology ; Isocitrate Lyase ; genetics ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; immunology ; microbiology ; Mice ; Nitric Oxide ; immunology ; Penicillium ; genetics ; immunology ; physiology ; Phagocytosis ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; omega-N-Methylarginine ; pharmacology

OBJECTIVElo investigate the effects of anti-VEGF antibody and anti-VEGF hairpin ribozyme gene on the proliferation, apoptosis and related gene expression of the leukemia K562 cells and the possible molecular mechanisms.

METHODSK562 cells were cultured in different concentrations of anti-VEGF antibody. The recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the vector-alone were introduced into K562 cells by lipofectamine mediation. Cell proliferative capacity was determined by MTT, colony formation assay and cells cycles analysis. Cell apoptosis was assayed by DNA ladder and Annexin V -FITC/PI flow cytometry.

RESULTSThe anti-VEGF antibody was able to inhibit growth and induce apoptosis of K562 cells in a dose-dependent manner. Exposure to anti-VEGF antibody at 0. 165 microg/ml for 72 hours, the cells exhibited typical DNA ladders. The apoptosis rate peaked at antibody concentration of 0. 825 microg/ml. RT-PCR showed a decrease of MRP and TOPO II expression but a relative constant expression of GST. The introduction of exogenous anti-VEGF ribozyme gene resulted in a decrease of the proliferative capacity and colony forming efficiency from (30.5 +/- 3.3) % in control group to (16.3 +/- 2.8) % in K562/RZ group, higher G1 and lower S ratio in cell cycle distribution in comparison with the control groups. Typical DNA fragmentation and higher Annexin V + ratio occurred in K562/RZ cells after treated with 0.5 micromot/L of As2O3 for 3 days, the apoptosis rate increased from 13.4% in control group to 31. 5% in As2O3 treated group.

CONCLUSIONAnti-VEGF antibody can inhibit growth, induce apoptosis and down-regulate the expression of MRP, TOPO II genes of K562 cells in vitro. Transfection with anti-VEGF ribozyme gene can inhibit the proliferation of the cells by delaying the progression G1 into S phase in cell cycles and induce cell apoptosis by down-regulating VEGF gene expression.

Antibodies ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Gene Expression Regulation ; Genetic Vectors ; Humans ; K562 Cells ; Liposomes ; RNA, Catalytic ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; immunology ; metabolism

Excessive production of nitric oxide (NO) and proinflammatory cytokines from activated microglia play an important role in human neurodegenerative disorders. Here, we investigated whether celastrol, which has been used as a potent anti-inflammatory and anti-oxidative agent in Chinese medicine, attenuates excessive production of NO and proinflammatory cytokines such as TNF-alpha and IL-1beta in LPS-stimulated BV-2 cells, a mouse microglial cell line. We report here that the LPS-elicited excessive production of NO, TNF-alpha, and IL-1beta in BV-2 cells was largely inhibited in the presence of celastrol, and the attenuation of inducible iNOS and these cytokines resulted from the reduced expression of mRNAs of iNOS and these cytokines, respectively. The molecular mechanisms that underlie celastrol-mediated attenuation were the inhibition of LPS-induced phosphorylation of MAPK/ERK1/2 and the DNA binding activity of NF-kappaB in BV-2 cells. The results indicate that celastrol effectively attenuated NO and proinflammatory cytokine production via the inhibition of ERK1/2 phosphorylation and NF-kappaB activation in LPS-activated microglia. Thus, celastrol may be an effective therapeutic candidate for use in the treatment of neurodegenerative human brain disorders.
Animals ; Cell Line ; Cytokines/*biosynthesis/drug effects ; Gene Expression Regulation, Enzymologic/drug effects/immunology ; Inflammation/immunology ; Inflammation Mediators/immunology ; Mice ; Microglia/*drug effects/immunology ; Mitogen-Activated Protein Kinases/*physiology ; NF-kappa B/metabolism/*physiology ; Nitric Oxide/*metabolism ; Nitric Oxide Synthase Type II/biosynthesis/drug effects ; RNA, Messenger/analysis ; Signal Transduction/*drug effects/physiology ; Transcription, Genetic/drug effects/immunology ; Triterpenes/*pharmacology

In order to investigate whether cytokine-induced killer (CIK) cells can induce apoptosis of bcr-abl(+) K562 cells, apoptosis of K562 cells and CEM cells induced by CIK cells, etoposide or camptothecin was detected with flow cytometry DNA assay. RT-PCR showed that K562 cells expressed the bcr-abl fusion gene, K 562 cells, K562 cells/etoposide or K562 cells/camptothecin groups showed no sub-G(1) peak. K562 cells/CIK cells group showed sub-G(1) peak (38.1%). CEM cells showed no sub-G(1) peak. CEM cells/camptothecin or CEM cells/etoposide groups showed sub-G(1) peak (23.5% or 32.3% respectively). CEM cells/CIK cells group showed sub-G(1) peak (45.4%). Etoposide or camptothecin did not induce apoptosis of K562 cells. CIK cells induce apoptosis of K562 cells. Bcr-abl fusion gene prevented apoptosis induced by etoposide or camptothecin, but did not prevent apoptosis induced by CIK cells. This property may support the observed adoptive immunologic effect of allogeneic bone marrow transplantation and donor lymphocyte transfusions of CML case relapsing after allogeneic bone marrow transplantation.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; immunology ; Camptothecin ; pharmacology ; Coculture Techniques ; Cytotoxicity, Immunologic ; Etoposide ; pharmacology ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; K562 Cells ; drug effects ; immunology ; metabolism ; Killer Cells, Lymphokine-Activated ; cytology ; immunology

Development of alternatively activated (M2) macrophage phenotypes is a complex process that is coordinately regulated by a plethora of pathways and factors. Here, we report that RBP-J, a DNA-binding protein that integrates signals from multiple pathways including the Notch pathway, is critically involved in polarization of M2 macrophages. Mice deficient in RBP-J in the myeloid compartment exhibited impaired M2 phenotypes in vivo in a chitin-induced model of M2 polarization. Consistent with the in vivo findings, M2 polarization was partially compromised in vitro in Rbpj-deficient macrophages as demonstrated by reduced expression of a subset of M2 effector molecules including arginase 1. Functionally, myeloid Rbpj deficiency impaired M2 effector functions including recruitment of eosinophils and suppression of T cell proliferation. Collectively, we have identified RBP-J as an essential regulator of differentiation and function of alternatively activated macrophages.
Animals ; Cell Polarity ; drug effects ; genetics ; immunology ; Cell Proliferation ; drug effects ; genetics ; Chitin ; immunology ; pharmacology ; Eosinophils ; cytology ; immunology ; Gene Expression Regulation ; drug effects ; immunology ; Immunoglobulin J Recombination Signal Sequence-Binding Protein ; genetics ; immunology ; Macrophage Activation ; drug effects ; genetics ; Macrophages ; cytology ; immunology ; Mice ; Mice, Transgenic ; T-Lymphocytes ; cytology ; immunology

The human colorectal carcinoma-associated GA733 antigen epithelial cell adhesion molecule (EpCAM) was initially described as a cell surface protein selectively expressed in some myeloid cancers. Gangliosides are sialic acid-containing glycosphingolipids involved in inflammation and oncogenesis. We have demonstrated that treatment with anti-EpCAM mAb and RAW264.7 cells significant inhibited the cell growth in SW620 cancer cells, but neither anti-EpCAM mAb nor RAW264.7 cells alone induced cytotoxicity. The relationship between ganglioside expression and the anti-cancer effects of anti-EpCAM mAb and RAW264.7 was investigated by high-performance thin-layer chromatography. The results demonstrated that expression of GM1 and GD1a significantly increased in the ability of anti-EpCAM to inhibit cell growth in SW620 cells. Anti-EpCAM mAb treatment increased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-alpha, caspase-3, cleaved caspase-3, and cleaved caspase-8 were unaltered. We observed that anti-EpCAM mAb significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. The expression of anti-apoptotic protein was inhibited by treatment with anti-EpCAM mAb, whereas the expression of pro-apoptotic proteins was increased. These results suggest that GD1a and GM1 were closely related to anticancer effects of anti-EpCAM mAb. In light of these results, further clinical investigation should be conducted on anti-EpCAM mAb to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.
Animals ; Antibodies, Monoclonal/*immunology/*therapeutic use ; Antigens, Neoplasm/*immunology ; Apoptosis/drug effects ; Cell Adhesion Molecules/*immunology ; Cell Line ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Colon/drug effects/immunology/metabolism/pathology ; Colonic Neoplasms/*drug therapy/genetics/*immunology/pathology ; Gangliosides/genetics/*immunology ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Male ; Mice ; Mice, Inbred BALB C

OBJECTIVETo investigate the expression of B7 co-stimulatory molecules in human multiple myeloma (MM) and the immunoregulatory effects of thalidomide on B7.1 co-stimulator.

METHODSThe immunoregulatory effects of thalidomide on the expression of B7-1 in human MM cell line was examined by detecting the changes in the expression of B7 co-stimulator on the cells using flow cytometry following the drug treatment.

RESULTSThe expression of B7.1 co-stimulator was lowly expressed in human MM cell line and MM patients, with a positivity rate of 0.8 and (2.19-/+2.13) for B7.1 and a rate of 26.4 and (30.28-/+28.11) for B7.2, respectively. Compared with the control group, the thalidomide-treated cells showed significantly increased percentage of CD-80 positive cells in a dose-dependent manner (but not at 0.1 microg/ml) (P<0.01), with the highest percentage reaching (17.7-/+1.53)% at thalidomide concentration of 5 microg/ml.

CONCLUSIONMM cells express low or undetectable levels of B7.1. Thalidomide can up-regulate the expression of B7-1 molecules on myeloma cells, which is probably one of the therapeutic mechanisms of thalidomide.

Adult ; Aged ; Aged, 80 and over ; B7-1 Antigen ; drug effects ; metabolism ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; drug therapy ; immunology ; pathology ; Thalidomide ; therapeutic use ; Up-Regulation ; drug effects

UNLABELLEDTo investigate the effect of sinomenine on the expression of chemokines and chemokine receptors of dendritic cells in patients with rheumatoid arthritis in vitro.

METHODSThe peripheral blood mononuclear cells isolated from 8 patients with rheumatoid arthritis were induced to differentiate into dendritic cells with GM-CSF and IL-4. The dendritic cells were exposed to sinomenine at high (5 mmol/L), moderate (2 mmol/L), and low (1 mmol/L) concentrations or treated with the control medium. The expression of CCR5 and CCR7 on the surface of the dendritic cells were measured by flow cytometry, and the CCR5 and CCR7 mRNA expressions were detected by semi-quantitative PCR. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expressions of CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (ITAC).

RESULTSCompared with the control cells, the dendritic cells treated with sinomenine, especially at high and moderate concentrations, showed significantly lowered mRNA and protein expressions of CCR5 and CCR7. Similar results were observed in the expressions of CXCL9 (MIG) and CXCL10 (IP-10), but not in CXCL11 (ITAC).

CONCLUSIONSinomenine produces therapeutic effect on rheumatoid arthritis possibly by inhibiting the expression of chemokines and chemokine receptors in the dendritic cells to suppress the chemotactic migration of the dendritic cells.

Adult ; Arthritis, Rheumatoid ; drug therapy ; immunology ; metabolism ; Chemokines ; genetics ; metabolism ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Middle Aged ; Morphinans ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; Receptors, Chemokine ; metabolism

Antigens ; immunology ; Antigens, Neoplasm ; drug effects ; immunology ; metabolism ; Gallbladder Neoplasms ; immunology ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Neoplastic Stem Cells ; immunology ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Tumor Cells, Cultured

OBJECTIVETo observe the effect of Bawei Xilei powder on CD3, CD4, CD8 T-lymphocytes in peripheral blood and colonic mucosa of rat with ulcerative colitis.

METHODSixty SD rats were randomly divided into 6 groups, normal group, model group, low, middle and high dosage Bawei Xilei powder group, Sulfasalazine group. Ulcerative colitis was induced by immunization with rabbit 's colonic mucous emulsified with completely Freund's adjuvant in all rats. Rats in low, middle and high dosage Bawei Xilei powder group were administered with 0.05, 0.1, 0.2 mg Bawei Xilei powder for 18 days by enema respectively. While rats in Sulfasalazine group were enema administered with 100 mg Sulfasalazine, and the rats in other group were administered with equal volume of saline enema as control. We analyzed expression of CD3, CD4, CD8 T-lymphocytes in peripheral blood by flow cytometry and in colonic mucous by immunohistochemistry.

RESULTIn peripheral blood, compared with normal group, in model group, the increased of CD4 T-lymphocytes and CD4 /CD8 ratio, the reduced of CD8 T-lymphocytes, these results were significant discrepancy (P < 0.01). Compared with model group, after treatment with Bawei Xilei powder, CD8 T-lymphocytes increased, but only high dosage Bawei Xilei powder group had discrepancy (P < 0.05). But low dosage Bawei Xilei powder group, other treatment groups' rats showed CD4/CD8 ratio were reduced significantly (P < 0.05). In colonic mucous, compared with normal group, in model group, Rats showed that expression of CD3, CD4 T-lymphocytes reduced and CD8 T-lymphocytes increased obviously (P < 0.05, P < 0.01). Compared with model group, expression of CD8 T-lymphocytes reduced significantly in all treatment groups (P < 0.05, P < 0.01).

CONCLUSIONBawei Xilei powder may regulate their balance between T-lymphocytes subgroup, consequently relieve inflammatory injury in favor of ulcer reparation and tissue regeneration.

Animals ; CD3 Complex ; metabolism ; CD4 Antigens ; metabolism ; CD8 Antigens ; metabolism ; Colitis, Ulcerative ; immunology ; metabolism ; Colon ; drug effects ; metabolism ; pathology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; Powders ; Rats ; T-Lymphocytes ; drug effects ; metabolism