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

This research was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on engraftment of hematopoietic stem cell in the sensitized recipient so as to provide the experimental evidence for the treatment of sensitized recipient's immune rejection after clinical allogeneic hematopoietic stem cell transplantation (HSCT). The BALB/c mice were divided into 4 groups: (1)mice sensitized on 7 day before transplant; (2)mice were sensitized on 7 day before transplant, and injected CTLA4Ig+anti-CD154 applied; (3)normal mice injected by corresponding isotype control IgG of CTLA4Ig and anti-CD154; (4)normal blank control mice. Each group had 15 mice. On day 0, mice of each group were irradiated lethally 8 Gy by linear accelerator, and the bone marrow cells of C57BL/6 labeled by fluorescence staining were injected via the tail vein. The fluorescent cell level in peripheral blood and organ tissue at different time points were detected by flow cytometry (FCM) for homing assessment. Survival rates and hematopoietic reconstitution were also monitored and recorded. The results showed that application of CTLA4Ig anti-CD154 could promote implantation of allogeneic HSC in sensitized recipients, induce the immune tolerance, prolong their survival time and accelerate the hematopoietic reconstitution within 28 days, compared with the sensitized group. It is concluded that applying CTLA4Ig and anti-CD154 can enhance the engraftment of HSCT and induce immune tolerance in the sensitized recipient after allogeneic HSCT and accelerate the hematopoietic reconstitution.
Abatacept ; Animals ; B7 Antigens ; antagonists & inhibitors ; CD28 Antigens ; antagonists & inhibitors ; CD40 Antigens ; antagonists & inhibitors ; CD40 Ligand ; antagonists & inhibitors ; Graft Rejection ; prevention & control ; Hematopoietic Stem Cell Transplantation ; Immune Tolerance ; Immunoconjugates ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Transplantation, Homologous

OBJECTIVETo investigate the anti-tumor activity of CCL21-exCD40L eukaryotic expression vector.

METHODSCCL21-exCD40L fusion gene were constructed by overlap PCR connecting CCL21 and exCD40L through a flexible linker (Gly3Ser)4, and then was cloned into expression vector pcDNA3.1(+). pcDNA3.1(+)/CCL21 and pcDNA3.1(+)/exCD were constructed as negative control. Wsestern blot was used to identify the fusion protein. CHO cells was transfected with pcDNA3.1(+)/CCL21-exCD, pcDNA3.1(+)/CCL21 and pcDNA3.1(+), respectively. The chemotatic function of the expressed product was detected by Transwell method and its anti-tumor activity was tested with vivo transfection.

RESULTSGene sequencing and restrictive digestion proved the successful construction of pcDNA3.1(+)/CCL21-exCD40L,and its expression was conformed by western blot. The transfectant supernantes of pcDNA3.1(+)/CCL21-exCD40 group had a significant chmotactic function to DCs, of which the cell numbers passing through the film was 14.95 times of blank control every high power microscope visual field. After tumor orthotoic injection of plasmid carrying fusion gene in Balb/c mouse, the tumor mass reduced remarkablely, and all the mouse in fusion gene group survived after 4 weeks.

CONCLUSIONCCL21-exCD40L fusion protein had a remarkable function to DCs and it can inhibit tumor growth and prolong the mouse survival time, which is more effective than all control group.

Animals ; CD40 Ligand ; genetics ; pharmacology ; CHO Cells ; Cell Line, Tumor ; Chemokine CCL21 ; genetics ; pharmacology ; Colonic Neoplasms ; therapy ; Cricetulus ; Dendritic Cells ; drug effects ; physiology ; Genetic Therapy ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology

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

OBJECTIVETo explore the effect of CD40 blockade in suppressing acute rejection of renal graft in rats.

METHODSWith Wistar rats as the donor and male SD rats as the recipients, rat models of acute renal graft rejection was established. The rat models were divided into therapy group and control group, and in the former group, CD40 ligand (CD40L) monoclonal antibody was injected daily for 4 consecutive days starting on the next day following kidney transplantation. On day 5 after the transplantation, the renal graft was harvested for histological examination, and graft rejection was evaluated semiquantitatively.

RESULTSThe mean semiquantitative score of the renal graft was 0.63∓0.52 in the therapy group, significantly lower than that of the control group (3.72∓1.48, P<0.05).

CONCLUSIONCD40L monoclonal antibody can inhibit acute renal graft in rats.

Animals ; Antibodies, Monoclonal ; pharmacology ; therapeutic use ; CD40 Antigens ; antagonists & inhibitors ; immunology ; CD40 Ligand ; immunology ; Female ; Graft Rejection ; drug therapy ; prevention & control ; Graft Survival ; drug effects ; Kidney Transplantation ; adverse effects ; Male ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar

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 analyze the cloning result of CD40 mutant from RPMI8226 cells, a multiple myeloma (MM) cell line, and study the change of the expressions of costimulatory molecules and the apoptosis of RPMI8226 cells after activated with CD40.

METHODSCD40 gene mutant in RPMI8226 cell was detected by RT-PCR and DNA sequencing. The cell lines were cultured with sCD40L, L929/CD40L, soluble 5C11 (an anti-CD40 mAb) plate-bound 5C11 and their respective controls. Their growth curves, change of phenotypes and cell cycles were detected. The signalosome of CD40 on RPMI8226 cells were analyzed with laser scanning confocal microscope.

RESULTSThere was a single base substitution (TCA-->TTA) in the open reading frame of CD40 from RPMI8226 cells, resulting in the conversion of a amino acid (Ser124Leu). Only plate-bound antibody could inhibit RPMI8226 cell proliferation [(2.5 +/- 0.6) x 10(5) vs (7.8 +/- 1.2) x 10(5), P <0.05] and cause G1 arrested [(58.0 +/- 3.6)% vs (42.0 +/- 2.3)%, P <0.05]. muCD40 was translocated to CD40 signalosome while CD40 activated.

CONCLUSIONThe mutated CD40 in RPMI8226 cell might decrease its affinity to CD40L, leading to the disorder of CD40 signal.

Antibodies, Monoclonal ; pharmacology ; CD40 Antigens ; genetics ; immunology ; CD40 Ligand ; genetics ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Mutational Analysis ; Humans ; Multiple Myeloma ; genetics ; pathology ; Mutation ; Phenotype ; Transgenes

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