OBJECTIVETo investigate the therapeutic effects of Qingre Quyu Granule (QQG) on the patients with severe carotid stenosis, and to explore the mechanism of it.

METHODSNinety-six patients with severe carotid stenosis were enrolled in the study and were classified into a QQG group (n=48) and a control group (n=48) randomly using consecutively numbered envelopes. The patients in the QQG group were given QQG and Western medicine, those in the control group were given Western medicine merely, the course of treatment was 16 weeks. All patients went through endarterectomy after treatment. Plaques were subjected to the analysis of CD3, CD68, soluble intercellular adhesion molecule 1 (ICAM-1), matrix metalloprotease-9 (MMP-9), CD40L, tenascin-C, and collagen content lipid content by immunohistochemistry or polarized light analysis.

RESULTSBy the end of experiment, the expressions of CD3, CD68, ICAM-1, MMP9, CD40L and tenascin-C on the plaques were statistically significant lower in the QQG group compared with the control group(P<0.01). The lipid content of the plaque was also significantly lower in the QQG group compared with the control group (P<0.01). The interstitial collagen in the tissue sections of the plaques was also significantly higher in the QQG group in comparison with the control group (P<0.01).

CONCLUSIONQQG could stabilize carotid artery plaques through inhibiting pro-inflammation factors and restraining the tenascin-C and MMP9 pathway.

Aged ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; CD3 Complex ; metabolism ; CD40 Ligand ; metabolism ; Carotid Arteries ; metabolism ; pathology ; Carotid Stenosis ; blood ; complications ; drug therapy ; Collagen ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Humans ; Immunohistochemistry ; Inflammation ; complications ; pathology ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipids ; blood ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Plaque, Atherosclerotic ; blood ; complications ; drug therapy ; Tenascin ; metabolism

CD40 Ligand ; pharmacology ; HL-60 Cells ; drug effects ; Humans ; fas Receptor ; metabolism

OBJECTIVETo observe the effects of Modified Liangge Powder (MLP) on the expressions of platelet toll like receptor 4 (TLR4) and the release of platelet-derived cytokines interleukin 8 (IL-8), beta platelet globulin (beta-TG), soluble CD40 ligand (sCD40L).

METHODSThe modulating effects on the release of cytokines from mice platelets by TLR4 ligand through monoclonal antibody blocking TLR4 on platelet were compared. The stimulated platelet by LPS was incubated with low (0.94 g/mL), medium (1.89 g/mL), and high (2.84 g/mL) dose of MLP contained serum. The changes of the platelet TLR4 expression and platelet-derived cytokines were observed.

RESULTSThe positive expression rate of platelet TLR4 obviously decreased (P < 0.01) and the release of sCD40L and beta-TG from platelets significantly increased (P < 0.01) after stimulated by LPS. However, the release of sCD40L and beta-TG from platelets obviously decreased by TLR4 monoclonal antibody (P < 0.05, P < 0.01). There was no statistical difference in IL-8 between before and after LPS stimulation (P > 0.05). Platelet TLR4 positive expression rate was significantly higher after incubated by medium and high doses of MLP contained serum (P < 0.01), and the releasing of sCD40L and beta-TG was lower in the serum contained groups. The inhibitory effects were enhanced in a dose-dependent manner.

CONCLUSIONSLPS induced platelet activation by TLR4 and released sCD40L and beta-TG, while the release of platelet IL-8 was not dependent on platelet TLR4-LPS pathway. MLP could inhibit LPS-stimulated sCD40L and beta-TG, inhibit the binding of platelet TLR4 and LPS in a dose-dependent manner, thus reducing the release of platelet cytokines.

Animals ; Beta-Globulins ; metabolism ; Blood Platelets ; drug effects ; metabolism ; CD40 Ligand ; metabolism ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Interleukin-8 ; metabolism ; Lipopolysaccharides ; adverse effects ; Male ; Mice ; Mice, Inbred ICR ; Serum ; Toll-Like Receptor 4 ; metabolism

The study was aimed to investigate the expression of Toll-like receptor 4 (TLR4) on platelets and to determine whether platelet TLR4 involves in its activation induced by lipopolysaccharide (LPS). Human platelet-rich plasma (PRP) and platelet suspension obtained from 15 healthy individuals pretreated with a concentration of 0.2 microg/ml of LPS in the presence or absence of thrombin (1 U/ml) for 1 hour. The expressions of TLR4, CD62P (P-select) and CD40L on platelets were detected by flow cytometry, and platelet TLR4 expression was further determined by Western blot analysis. The results indicated that the percentage of TLR4-positive platelets induced by thrombin was increased by 32.34% compared with the resting platelets (25.44%, p < 0.05). TLR4 expression on platelets treated with LPS was remarkably elevated in the presence or absence of thrombin. However, the expression level of the former was much higher than that of the latter and thrombin stimulation alone (p < 0.05). Moreover, the similar results were found in Western blot analysis. Synchronously, expressions of CD62P and CD40L on resting platelets were 6.39% and 2.45%, they were also markedly increased when treated with thrombin (42.68% and 14.8%) and LPS respectively, and the increases of expression of CD62P and CD40L were more significant when stimulated with both LPS and thrombin (63.03% and 13.94%). Although anti-TLR4 antibody inhibited significantly the increase of TLR4, CD62P and CD40L on platelets induced by LPS, which did not affect their increase induced by thrombin. In conclusion, the evidence has been shown that functional TLR4 can be expressed on human platelets. It may involve in platelet activation as an important mediator of LPS-induced CD62P and CD40L expressions on platelets.
Adult ; Blood Platelets ; metabolism ; CD40 Ligand ; genetics ; Humans ; Lipopolysaccharides ; pharmacology ; Middle Aged ; P-Selectin ; metabolism ; Platelet Activation ; Toll-Like Receptor 4 ; metabolism

Pancreatic islet transplantation can correct the abnormal glucose metabolism of Type 1 diabetes. Although immunosuppressants greatly reduce the acute rejection rate in transplant patients, the long-term side effects can be debilitating. Therefore, researchers are seeking to develop new immunosuppressive regimens that induce maximal levels of immunosuppression with minor side effects. Rosmarinic acid (Ros A) is a secondary metabolite of certain herbs and has multiple biological activities, including anti-inflammatory effects. Here, we have investigated whether treatment of mice with a combination of Ros A and anti-CD154 monoclonal antibody (MR1) improves islet allograft survival in a murine model. After transplantation, the mice were treated with either Ros A, MR1, or both (the "double" treatment). Allograft survival was prolonged in the double-treated animals compared to animals that received only Ros A or MR1. As is the case with the single-treated animals at 15 days after transplantation, the double-treated recipients did not display a significant decrease in the expression of cytokines or the population of activated T cells. Infiltrating CD3+ T cells were reduced in the MR1- or double therapy relative to control or RosA group. However, at the same time point, double-treated graft showed fewer apoptotic cells and increased expression of insulin and glucagons, compared to the single-treatment groups. Furthermore, long-term (>150 days) allografts that were received with double therapy exhibited larger islet clusters and contained more insulin- and glucagon-positive cells, relative to the MR1-treated grafts. In conclusion, treatment with both Ros A and MR1 has a synergistic effect in murine islet allotransplantation.
Animals ; Antibodies, Monoclonal/*pharmacology ; Apoptosis/drug effects ; CD40 Ligand/*immunology ; Cinnamates/*pharmacology ; Cytokines/biosynthesis ; Depsides/*pharmacology ; Diabetes Mellitus, Experimental ; Flow Cytometry ; Glucose/metabolism ; Glucose Tolerance Test ; Graft Survival/*drug effects ; In Situ Nick-End Labeling ; Injections, Intraperitoneal ; Islets of Langerhans/drug effects/pathology ; *Islets of Langerhans Transplantation ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Time Factors ; Transplantation, Homologous

OBJECTIVETo investigate the transduction efficiency of serotype 1, 2, 5, 6, 7, 8, 9, 10 recombinant adeno-associated viruses (rAAV) in human lung cancer cell line A549 cells and compare the transduction efficiency of conventional AAV vectors with that of self-complementary AAV (scAAV) vectors. Furthermore, the capacity of A549 cells expressing transgenic CD40L to stimulate dendritic cells (DCs) was evaluated.

METHODSLung cancer A549 cells were infected with 1 x 10(4) particules per cell of AAV encoding the green fluorescent protein (GFP) or human CD40L driven by CMV promotor, and transgene expression was analyzed by flow cytometry and fluorescence microscopy. Stimulation of isolated human dendritic cells by CD40L-expressing tumor cells was quantified by measuring secreted interleukin-12 with immunoassay.

RESULTSSerotype AAV2/5 transduced A549 cells much more efficiently than serotypes AAV2/1, AAV2/2, AAV2/6, AAV2/7, AAV2/8, AAV2/9 and AAV2/10. The transduction efficiency of scAAV2/5 was significantly higher than that of conventional AAV2/5. Furthermore, pre-treatment with carboplatin substantially increased AAV-mediated transgene expression. The scAAV2/5 vectors encoding human CD40L was used to generate CD40L. A549 cells transduce by these vectors were co-cultured with immature human DCs. As a consequence, interleukin-12 was released and measured in the culture supernatant. Specificity of immunostimulatory effect of CD40L was confirmed by blocking with a monoclonal antibody binding to human CD40L.

CONCLUSIONscAAV2/5 transduce lung adenocarcinoma A549 cell efficiently, and co-administration of chemotherapeutic agent carboplatin further enhances its transduction efficiency. It is confirmed that lung cancer cells infected with a CD40L-encoding scAAV2/5 construct can activate human DCs to secrete interleukin-12. Our findings provided a basis for future immunotherapeutic approaches including intratumoral transfer of stimulating factors.

Antineoplastic Agents ; pharmacology ; Blotting, Western ; CD40 Ligand ; genetics ; metabolism ; physiology ; Carboplatin ; pharmacology ; Cell Line ; Cell Line, Tumor ; Coculture Techniques ; Dendritic Cells ; cytology ; secretion ; Dependovirus ; classification ; genetics ; Flow Cytometry ; Gene Expression ; drug effects ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Immunoassay ; methods ; Interleukin-12 ; secretion ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism ; Serotyping ; Transfection

OBJECTIVETo investigate the effect of prolactin on CD40 and CD154 expressions on the surface of peripheral blood mononuclear cells in patients with systemic lupus erythematosus (SLE) and explore the role of prolactin in the pathogenesis of SLE.

METHODSThe serum prolectin level was detected in 30 SLE patients and 20 healthy volunteers, from whom peripheral blood mononuclear cells (PBMCs) were also isolated to examine the expressions of CD40 and CD154 using flow cytometry.

RESULTSCD154 significantly increased on the PBMCs in SLE patients with high serum prolectin level in comparison with that in patients with normal prolactin level or the normal controls (P<0.05). When the PBMCs were incubated with recombinant human prolactin, CD154 expression was significantly increased in SLE patients with normal serum prolactin level (P<0.05), but not in the normal control group (P>0.05). Incubation of the PBMCs in the presence of bromocriptine did not result in significantly decreased CD154 expression in SLE patients irrespective of the prolactin level, nor was significant difference found in CD40 expression on the surface of PBMCs between SLE patients and the normal controls(P>0.05).

CONCLUSIONProlactin plays an important role in the pathogenesis of SLE by increasing CD154 expression on the PBMCs, and bromocriptine produces no significant inhibitory effect on either endogenous or exogenous prolectin.

Adult ; CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Case-Control Studies ; Female ; Gene Expression Regulation ; drug effects ; Humans ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lupus Erythematosus, Systemic ; blood ; metabolism ; pathology ; Male ; Prolactin ; blood ; pharmacology

Cytokine and chemokine receptors play a key role in inflammation caused by rheumatoid arthritis (RA). Two isoforms of human CC chemokine receptor R2 (CCR2), the receptor of monocyte chemoattractant protein 1 (MCP-1), have been identified but their relative expression in fibroblast-like synoviocytes (FLS) and their contribution to inflammatory responses mediated by MCP-1 or inflammatory cytokines in patients with RA remain uncertain. We examined the pattern of expression of two CCR2 isoforms upon stimulation by proinflammatory cytokines and CD40 ligation. FLS were prepared from the synovial tissues of RA patients and cultured in the presence of MCP-1, soluble CD40 ligand (sCD40L), TGF-beta, IL-1beta, IL-18, IL-15, and LPS. CCR2A and CCR2B expression was examined by immunohistochemistry, RT-PCR and western blot analysis. IL-15, TNF-alpha and MCP-1 production was determined by ELISA. Immunohistochemistry showed that CCR2A is highly expressed in RA synovium compared with OA synovium. Transcripts of both CCR2A and CCR2B were detected in FLS. Exogenous MCP-1, CD40L, TGF-beta, and IL-15 significantly increased the expression of CCR2A but not CCR2B. Exposure of FLS to sCD40L caused strong upregulation of CCR2A but not of CCR2B protein expression. MCP-1 increased the proliferation of FLS and the production of IL-15, TNF-alpha, and IL-18. Because CCR2A is the main target of regulation by cytokines and CD40 ligation, the relatively higher expression of CCR2A on the cell surface suggests that this isoform of MCP-1 receptor functions as the principal mediator of inflammatory signals in RA FLS.
Arthritis, Rheumatoid/*metabolism ; CD40 Ligand/*pharmacology ; Cells, Cultured ; Chemokine CCL2/*pharmacology ; Chemokines/biosynthesis ; Fibroblasts/*metabolism ; Humans ; Protein Isoforms ; Receptors, CCR2/*biosynthesis ; Synovial Membrane/*pathology ; Transforming Growth Factor beta/*pharmacology

OBJECTIVETo investigate the effects of simvastatin on vascular cell adhesion molecule-1 (VCAM-1) expression and adhesive function in ECV-304 cells treated with CD40L.

METHODSHuman umbilical vein endothelial cell (HUVEC) was cultured and treated with various concentrations CD40L alone or in combination with various concentrations simvastatin in the absence or presence of mevalonic acid (400 micromol/L). RT-PCR and FCM analysis were used to determine VCAM-1 expression and lymphocytes adhesion to endothelial cells.

RESULTSSimvastatin (0 - 10 micromol/L) decreased in a concentration-dependent manner the expression of VCAM-1 induced by CD40L and this effect could be blocked by cotreatment with mevalonic acid. Moreover, Simvastatin also significantly decreased adhesion capacity of lymphocytes to endothelial cells induced by CD40L.

CONCLUSIONSimvastatin downregulates VCAM-1 expression and adhesive capacity of lymphocytes to endothelial cells induced by CD40L.

CD40 Ligand ; metabolism ; Cell Adhesion ; Cells, Cultured ; Endothelium ; drug effects ; metabolism ; Endothelium, Vascular ; cytology ; Humans ; RNA, Messenger ; metabolism ; Simvastatin ; pharmacology ; Umbilical Veins ; cytology ; Vascular Cell Adhesion Molecule-1 ; metabolism

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low)and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low)T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two-signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.
Transplantation, Homologous ; T-Lymphocytes, Regulatory/cytology/immunology ; Skin Transplantation/*immunology ; Signal Transduction/drug effects/immunology ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Male ; Lymphocyte Depletion ; Lymphocyte Activation/immunology ; Interleukin-4/biosynthesis ; Interleukin-10/biosynthesis ; Graft Rejection/*immunology/prevention & control ; Flow Cytometry ; Cytotoxicity, Immunologic/immunology ; CD8-Positive T-Lymphocytes/cytology/immunology/metabolism ; CD40 Ligand/*immunology ; CD4-Positive T-Lymphocytes/cytology/immunology/metabolism ; Antigens, CD45/*immunology ; Antigens, CD4/*immunology ; Antibodies, Monoclonal/administration & dosage/*pharmacology ; Antibodies, Blocking/administration & dosage/pharmacology ; Animals