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

OBJECTIVETo study and compare the anti-inflammatory effect and molecular mechanism of artemisinin and dihydroartemisinin.

METHODMouse mononuclear macrophage RAW264.7 cells were stimulated to release inflammatory mediators such as TNF-alpha, IL-6 and NO, in order to assess the drugs' inhibitory effect on macrophage's release of above inflammatory mediators. The levels of TNF-alpha and IL-6 were determined by ELISA and the cytotoxicity was determined by MTT method. The protein expression of iNOS, COX-2 and beta-actin were tested by Western blot. The enzymatic activity of COX-2 was determined by colorimetric method.

RESULTDihydroartemisinin significantly inhibited LPS-induced release of TNF-alpha, IL-6 and NO from RAW264.7 in mice with the concentration range of 12.5 - 100 micromol x L(-1), and showed good dose dependence. Artemisinin only inhibited the IL-6 release to a certain extent.

CONCLUSIONDihydroartemisinin inhibits macrophages from releasing inflammatory factors TNF-alpha and IL-6 and inflammatory mediators NO by down-regulating iNOS protein. Artemisinin may help dihydroartemisinin to show its anti-inflammatory effect through metabolism.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Artemisinins ; pharmacology ; Cell Line ; Gene Expression ; drug effects ; Inflammation Mediators ; immunology ; Interleukin-6 ; genetics ; immunology ; Macrophages ; drug effects ; immunology ; Mice ; Nitric Oxide ; immunology ; Tumor Necrosis Factor-alpha ; genetics ; immunology

OBJECTIVETo elucidate the gene regulatory pattern of Epimedium flavonoids (EF) in immune homeostasis remodeling in the aged rats.

METHODS(1) To quantitatively analyse the apoptosis percentage of lymphocyte in spleen of aged, young and EF treated rats using flow cytometry. (2) To analyse the lymphocyte gene expression profiles of different groups using gene chips (Rat Genome U34A).

RESULTS(1) Detection of lymphocyte apoptosis percentage showed that there was significant difference in comparing between aged group and young group, between EF treated group and aged group (P < 0.01). (2) As compared with that in the young group, in the aged group, 116 genes were up-regulated and 215 down-regulated. As compared with that in the old group, in the EF treated group, 447 genes were up-regulated and 456 down-regulated, which involved the aspects as cell apoptosis and cell proliferation regulation, etc.

CONCLUSION(1) The expression pattern characterized by up-regulation of apoptosis promoting genes expression and down-regulation of apoptosis inhibiting genes expression, is the important gene background of immuno-homeostasis imbalance in the aged. (2) The role of EF is to reverse the abnormal changes of gene expressions with opposite functions, i.e. the apoptosis promoting and inhibiting, proliferation enhancing and antagonizing genes, to reconstruct a beneficial equilibrium of gene expression and thus to further remodel the immuno-homeostasis in the aged.

Adjuvants, Immunologic ; pharmacology ; Aging ; Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epimedium ; chemistry ; Female ; Flavonoids ; pharmacology ; Gene Expression ; Gene Expression Profiling ; Homeostasis ; Lymphocytes ; immunology ; metabolism ; Male ; Neuroimmunomodulation ; drug effects ; Rats ; Rats, Sprague-Dawley ; T-Lymphocytes ; immunology ; metabolism

OBJECTIVETo study the influence of SGHWJN particles on inflammation and the mediators of inflammation in esophageal tissues of rat with reflux esophagitis.

METHODFifty SD rats were randomly divided into 5 groups, namely, a control group, a sham-operated group, a model group, a SGHWJN particles group and a PPI group. Reflux esophagitis was induced by adopting partial pyloric ligation plus cardiomyotomy. One week later, the rats were orally administered twice daily for 28 days. Pathological changes of esophagus mucous membrane were evaluated by using HE staining and Harry S. Cooper's method in every groups. MDA and SOD contents in esophageal tissues were measured by colorimetric method. Expression of TNF-alpha in esophageal tissues were examined by real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (RT-FQ-PCR) with SYBR Green.

RESULTModel group, esophageal inflammation scores, expression of TNF-alpha in esophageal tissues and MDA contents compared with the normal group and sham operation group were significantly higher (P < 0.05). SOD contents in the esophageal tissues of the model group was significantly lower than that of control group and sham-operated group (P < 0.05). SGHWJN particles group and PPI group of esophageal tissue inflammation scores, expression of TNF-a in esophageal tissues and MDA levels than those in model group decreased significantly (P < 0.05). SOD content was significantly higher than that of model group (P < 0.05), SGHWJN particles group and PPI group showed no statistically significant difference between the above-mentioned indicators. The above-mentioned indicators showed no statistically significant difference between the normal group and sham-operated group. MDA content and expression of TNF-alpha in esophageal tissue was positively correlated with inflammatory scores of model group (r = 0.813). Model group esophageal tissue SOD content and inflammation scores were negatively correlated (r = -0.847). Esophageal tissue SOD levels were negatively correlated with MDA levels (r = -0.863).

CONCLUSIONSGHWJN particles can effectively inhibit inflammation in rat with reflux esophagitis through regulating TNF-alpha, SOD and MDA.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Esophagitis, Peptic ; drug therapy ; genetics ; immunology ; Esophagus ; drug effects ; immunology ; Female ; Gene Expression ; drug effects ; Humans ; Inflammation Mediators ; immunology ; Male ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; genetics ; immunology

OBJECTIVETo elucidate the immunomodulatory mechanism of phenylethanoid glycosides from the seeds of Plantago asiatica by testing its effects on the maturing of murine bone marrow derived dendritic cells (DCs).

METHODMonocytes generated from bone marrow of Balb/cj mouse were cultured for 6 days in complete RPMI 1640 medium containing 10% FBS, rmGM-CSF and rmIL-4.50 mg x L(-1) acteoside or isoacteoside was added to cells on day 6 of culture for 24 h. The surface molecules expression level of DCs and their phagocytose ability were analysis by flow cytometry.

RESULTBoth acteoside and isoacteoside could increase the expression of CD11c, CD86, MHC II and CD80 on DCs surface. The ability of unstimulated DCs to uptake FITC-dextran was higher than that of phenylethanoid glycosides or LPS treated DCs.

CONCLUSIONBoth acteoside and isoacteoside could induce maturation of murine dendritic cells.

Animals ; B7-1 Antigen ; immunology ; metabolism ; B7-2 Antigen ; genetics ; immunology ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; Gene Expression ; drug effects ; Glycosides ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Phagocytosis ; drug effects ; Plant Extracts ; pharmacology ; Plantago ; chemistry ; Seeds ; chemistry

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

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

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

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 observe the efficacy of herbal cake-separated moxibustion on the expression of erythrocyte CD58 in different ages of healthy people and explore the differences of the therapeutic effect in different ages and its mechanism.

METHODSA total of 82 health participants were divided into a young age group and a middle-old age group according to the ages. They were treated with herbal cake-separated moxibustion on Shenque (CV 8), Guanyuan (CV 4), Zusanli (ST 36), Pishu (BL 20), Shenshu (BL 23) with cake made by Shudihuang (Radiz Re hmanniae Preparata), Shanyao (Rhizoma Dioscoreae), Shanzhuyu (Fructus Corni ), etc. The treatment was given for 10 sessions once other day and each acupoint for 3 successive dosages. The mean fluorescence intensities of erythrocyte CD58 were measured by flow cytometry before and after moxibustion.

RESULTSAfter moxibustion, erythrocyte CD58 expression were significantly higher than that before moxibustion in two groups (both P < 0.01), particularly in young age group, which was significantly higher than that in middle-old age group (P < 0.01).

CONCLUSIONThe effect of moxibustion in youth is evidently superior to that in middle-old age. Its mechanism is connected with that moxibustion can enhance the expression of erythrocyte CD58.

Acupuncture Points ; Adult ; Age Factors ; CD58 Antigens ; genetics ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Erythrocytes ; drug effects ; immunology ; Female ; Gene Expression ; drug effects ; Humans ; Immunity ; drug effects ; Male ; Middle Aged ; Moxibustion ; Young Adult