As CD40 transduces activation signals involved in inflammatory and immune disorders, we explored the expression and response to CD40 engagement in human glioma cell lines in this study. The CD40 expression in BT-325 and U251 cells was flow cytometrically detected. The cells were incubated with srhCD40L for 72 h to assess its effects on cell growth in vitro. TNF-α expression was quantified by real-time PCR, and protein expression was analyzed by ELISA. The I-κb mRNA was detected by RT-PCR. I-κB expression decreased after stimulation with 1 μg/mL srhCD40L, but it was upregulated after the cells were pretreated with CD40 antibody. srhCD40L significantly inhibited the proliferation of the CD40+ human glioma cells. The stimulation of CD40+ glioma cells with soluble CD40L (CD154) up-regulated the expression of TNF-α at both mRNA and protein levels. We are led to conclude that CD40L/CD40 could inhibit human glioma cells through I-κb signaling pathway. Interferon-γ can augment CD40 expression and the inhibitory effect of CD40 ligand on cell growth in vitro. These results suggest that srhCD40L may benefit the therapy strategy of glioma.
CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Cell Proliferation ; Cells, Cultured ; Glioma ; genetics ; metabolism ; pathology ; Humans ; NF-kappa B ; metabolism ; Signal Transduction ; physiology ; Tumor Cells, Cultured

The interaction of CD40 with its cognate ligand, CD40L (CD154), plays important roles in immune responses. Blockade of CD40-CD40L signal pathway can protect the progression of antibody- and cell-mediated autoimmune diseases, and reduce allograft rejection thus prolonging graft survival, even engendering long-lived antigen-specific tolerance. The present study aims to enhance the binding activity of CD40 by incorporating an isoleucine zipper (IZ) trimeric motif into CD40 ectodomain to promote the formation of soluble CD40 trimers, which would be useful for blocking CD40-CD40L interaction. A prokaryotic expression vector for soluble human CD40 ectodomain fused with an IZ motif and a hexa-histidine (His6) tag at its carboxyl terminus (sCD40IZ) was constructed by multiple round PCR using cloned CD40 cDNA as a template. The recombinant sCD40IZ protein was expressed highly in Escherichia coli (E. coli) with a molecular weight of 27kD, which is consistent with its theoretical value. It mainly existed in inclusion bodies. After refolding from inclusion bodies, soluble sCD40IZ protein was purified by gel filtration. Its molecular weight in solution was about 91kD when determined by gel filtration, suggesting that it most probably existed in the form of trimers. Moreover, this protein could bind to CD40L expressed on Jurkat T cells and its binding activity was significantly higher than that of soluble CD40 without an IZ motif. These results suggest that incorporation of an IZ motif at the carboxyl terminus of soluble CD40 can facilitate the formation of trimers and enhance its binding activity with CD40L. Thus, the trimeric CD40 protein may be used to block CD40-CD40L signal pathway, suggesting that it may have potential application in preventing autoimmune diseases and transplantation rejection.
CD40 Antigens ; biosynthesis ; genetics ; metabolism ; CD40 Ligand ; metabolism ; Escherichia coli ; genetics ; metabolism ; Humans ; Isoleucine ; biosynthesis ; genetics ; Leucine Zippers ; genetics ; Protein Multimerization ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism

OBJECTIVETo investigate the expression levels of CD40, sCD40L, hs-CRP, WBC in acute coronary syndrome (ACS) patients and the association between CD40-1C/T single nucleotide polymorphism and risk of ACS in Han Chinese, moreover, the regulatory effects of IFN-gamma and fluvastatin on the expression of CD40 in peripheral blood mononuclear cell (PBMNC) were also observed.

METHODS(1) 160 ACS patients and 92 control patients diagnosed by coronary angiography were recruited. Enzyme linked immunosorbent assay, particle enhanced immunoturbidimetric assay, flow cytometry were used to detect the levels of soluble CD40L, hs-CRP, and WBC count. (2) The CD40 genotype and allele frequency were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing technology. (3) PBMNC were separated by density gradient centrifugation heparinized venous blood from 40 ACS patients, cultured for 24 h with or without 100 ng/ml IFN-gamma in the absence or present of 10 micromol/L fluvastatin. The CD40 expression levels were then detected by flow cytometry.

RESULTSInflammatory cytokine CD40, sCD40L, hs-CRP levels were significantly higher in ACS patients than in controls. The CD40-1C allele frequency was 0.606 in ACS group and 0.489 in controls, while T allele frequency was 0.394 in ACS group and 0.511 in controls. The frequency of CC genotype was significantly higher in ACS group than in controls (P < 0.01). C allele carriers had significantly higher risk of ACS (OR = 1.608, 95%CI: 1.12 - 2.32, P = 0.011). CD40 production increased after 24 h culturing and the CD40 levels were significantly higher in subjects with CC genotype than that in subjects with CT or TT genotype [CC: (14.78 +/- 4.56) MFI, CT: (11.98 +/- 4.12) MFI, TT: (9.86 +/- 3.83) MFI, P < 0.05]. IFN-gamma further increased PBMNC CD40 expressions in all subjects after culturing for 24 h and fluvastatin equally inhibited IFN-gamma induced PBMNC CD40 expression from various genotypes (CC, CT, TT was 30.3%, 26.3%, 29.3% respectively, all P > 0.05).

CONCLUSIONInflammatory cytokines were increased in ACS patients and CD40-1C/T polymorphism is associated with higher risk for ACS in Han Chinese.

Acute Coronary Syndrome ; genetics ; Aged ; CD40 Antigens ; genetics ; metabolism ; CD40 Ligand ; genetics ; metabolism ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide

OBJECTIVE: To investigate the expression of costimulatory molecules CD28/B7 and CD40/CD40L in T and B lymphocytes as well as its relations with total IgE (TIgE), eosinophil cationic protein (ECP) in serum and nasal allergic symptoms in patients with allergic rhinitis (AR). The effect of specific immunotherapy (SIT) on them were also investigated. METHOD: Thirty allergic allergic rhinitis patients were chosen as observation group, and 30 healthy patients as control group. Cytofluorometric analysis was used to compare the expression level of CD28/B7-1, B7-2 and CD40/CD40L on T cells and B cells in the two groups. The relationship between the CD28/ B7-1, B7-2 and CD40/CD40L expression level and serum Total IgE, ECP level were analyzed. RESULT: The expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells in allergic rhinitis patients were significantly higher than in the healthy, and serum level of TIgE has a positive relationship with the expression level of CD40L on T cells. ECP has a positive relationship with the expression level of B7-2 on B cells. The expression level of B7-1 showed no significant difference between the two groups. After specific immunotherapy for 6 months, the expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells were decreased in allergic rhinitis patients but still higher than in healthy. CONCLUSIONS The upregulated level of costimulatory molecules CD28/B7-2 and CD40/ CD40L on T cells and B cells may play an important role in the pathogenesis of allergic rhinitis, specific immunotherapy can downregulate the expression level of CD28/B7-2 and CD40/CD40L, and decrease the serum level of TIgE, it may be a possible mechanism in the treatment of allergic rhinitis.
Adult ; B-Lymphocytes ; immunology ; metabolism ; B7-2 Antigen ; metabolism ; CD28 Antigens ; metabolism ; CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Case-Control Studies ; Desensitization, Immunologic ; Female ; Humans ; Immunoglobulin E ; blood ; Male ; Rhinitis, Allergic, Perennial ; blood ; immunology ; metabolism ; therapy ; T-Lymphocytes ; immunology ; metabolism

CD40/CD40L is a pair of complementary transmembrane glycoproteins, expressed on immune cells, endothelial cells, smooth muscle cells, platelets and other cells involved in regulation of immunity, inflammation, coagulation and other pathophysiologic states. A large number of researches have demonstrated that, when atherosclerosis occurs, CD40L ligates CD40; subsequently CD40 is activated and stimulates downstream signaling pathways, including nuclear factor-kappaB, with consequent up-regulation of proinflammatory and proatherogenic genes. Thus it plays an important role in the occurrence, development and plaque-rupture of atherosclerosis. CD40/CD40L is a bridge between immunity, inflammation, and a hypercoagulable state, and may be an important target for prevention and treatment of cardiovascular disease.
Animals ; Atherosclerosis ; genetics ; physiopathology ; CD40 Antigens ; genetics ; CD40 Ligand ; genetics ; Humans ; Immunity ; physiology ; Inflammation ; physiopathology ; NF-kappa B ; metabolism ; Polymorphism, Genetic ; Thrombophilia ; physiopathology

OBJECTIVE: To investigate the expression of inflammatory molecule CD40 in the pallium and hippocampus of rats after status epilepticus (SE).
 METHODS: The expression of CD40 in the pallium, the different areas of hippocampus and the different cells from the lithium-pilocarpine epileptic rats at different time points were examined by immunohistochemistry and double-immunofluorescent labeling.
 RESULTS: After SE, CD40 expression was obviously inhibited, especially in hippocampus. CD40 was mainly expressed in the activated microglia. CD40 positive cells reached a peak at the 3rd day and returned to a slightly higher level at the 7th day after SE compared with the level before SE.
 CONCLUSION Elevation of CD40 expression in the activated microglia can promote inflammatory injury of rat's hippocampus, suggesting that CD40 induced-signal pathway is involved in inflammatory injury in the hippocampus after SE.
Animals ; CD40 Antigens ; metabolism ; Epilepsy ; Hippocampus ; metabolism ; Immunohistochemistry ; Lithium ; Microglia ; metabolism ; Pilocarpine ; Rats ; Rats, Sprague-Dawley ; Status Epilepticus

Apoptosis is responsible for the loss of thyrocytes in autoimmune thyroiditis. Recent investigations into the pathogenesis of apoptosis have revealed that the important roles of suicide molecules expression on both thyrocytes and cytotoxic T-lymphocytes. To study the mechanism of thyrocyte loss in various forms of thyroiditis, we evaluated in situ expression patterns of CD40, Fas, and Fas-L on thyrocytes and infiltrating inflammatory cells by immunohistochemical staining of thyroid samples obtained from 49 patients (Graves' disease, n=10 : Hashimoto's thyroiditis, n=14; nonspecific lymphocytic thyroiditis, n=11; subacute granulomatous thyroiditis, n=11; normal, n=3). The role of cytotoxic T-lymphocytes was also evaluated by analyzing the expression of granzyme B along with their phenotypic characteristics. CD40 was not expressed on thyrocytes of normal controls while they showed a diffuse expression of Fas and a scattered focal expression of Fas-L. The plump thyrocytes proximal to the inflammatory infiltrates showed more intense expressions of these three molecules in various forms of thyroiditis and a close correlation was found between CD40 and Fas-L expression on thyrocytes. Unlike Fas, which was expressed on infiltrating lymphocytes in all groups, Fas-L was not expressed on infiltrating lymphocytes, except those in subacute granulomatous thyroiditis. Granzyme B expressing activated cytotoxic T-lymphocytes occupied a negligible proportion of CD8+ T-lymphocytes in various forms of thyroiditis, and no difference was found in terms of their proportions according to the type of thyroiditis. These results show the acquisition of CD40, Fas and Fas-L molecules on thyrocytes proximal to inflammatory cell aggregates and the negligible expression of granzyme B and Fas-L on the infiltrating lymphocytes, and suggest that Fas and Fas-L mediated apoptosis of thyrocytes (fratricide) may be more important than T cell-mediated cytotoxicity in various forms of thyroiditis.
Antigens, CD40/*metabolism ; Antigens, CD95/metabolism ; Apoptosis/*physiology ; Graves' Disease/*metabolism/pathology ; Human ; Membrane Glycoproteins/metabolism ; Reference Values ; Thyroiditis, Autoimmune/*metabolism/pathology

This study was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice', and provide the evidences of acquired immune tolerance for allogeneic bone marrow transplantation. The mice sensitized on 7 day before transplant were divided into 4 groups: (1)CTLA4Ig+ anti-CD154 isotype control IgG; (2)anti-CD154 +CTLA4Ig isotype control IgG; (3)CTLA4Ig and anti-CD154; (4)isotype control IgG of CTLA4Ig and anti-CD154. CTLA4Ig and anti-CD154 used in normal BALB/c mice as isotype control IgG. Each mouse in all groups received CTLA4Ig and anti-CD154 (or corresponding isotype control IgG) 500 µg respectively, and was injected via tail vein on 7 day before transplant. There were 5 mice in each group. The mice were sacrificed on day 0, then the number of CD19(+)CD69(+)B cells, CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- T cells were measured by flow cytometry. Changes of cytokines and sensitized antibody were tested by ELISA or flow cytometry. The results showed that the numbers of CD19(+)CD69(+)B cells were significantly increased in comparison with the normal group (P < 0.01) , whereas the numbers of cells were significantly decreased when blocking B7/CD28 or /and CD40/CD154 co-stimulatory signals (P < 0.01) . Blocking these 2 signals together displayed a synergistic effect (P < 0.01) . The central memory and effector T cells were defined as CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- respectively, those increased significantly after sensitized in comparison with those in normal group, whereas their numbers decreased when blocking B7/CD28 or/and CD40/CD154 co-stimulatory signals. Blocking these two signals together, displayed a synergistic effect (P < 0.01). Cytokines, IgG and IgM in all groups were not significantly different. Sensitizing antibody test showed that the fluorescence intensity of sensitized group significantly increased as compared with normal group, whereas fluorescence intensity of CTLA4Ig or/and anti-CD154 treated groups significantly decreased as compared with sensitized group (P < 0.01) . It is concluded that blocking the B7/CD28 or/and CD40/CD154 co-stimulatory signal can inhibit the cellular and humoral immune function, whereas blocking these two signals together displays a synergistic effect.
Animals ; B7-1 Antigen ; metabolism ; Bone Marrow Transplantation ; CD28 Antigens ; metabolism ; CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Immune Tolerance ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Signal Transduction ; Transplantation, Homologous

Impaired regulation of apoptosis is known to be associated with the development of various cancers, and Fas/Fas-ligand (FasL) is known to play an important role in apoptosis. CD40 is a cell surface receptor, which when ligated modulates apoptosis in some cell types. The expressions of CD40 and FasL were examined in 10 normal skins, 7 Bowen's disease skins, 10 squamous cell carcinomas (SCCs) and 12 basal cell carcinomas (BCCs) immunohistochemically. In the normal epidermis, CD40 was more highly expressed in the keratinocytes of the squamous cell and granular layers than in those of the basal layer, and FasL expression was observed in the cell membrane of keratinocytes at the basal and squamous cell layers. CD40 expression was significantly higher in SCCs than in normal or Bowen's disease skin, while FasL expression was significantly higher in Bowen's disease than in SCCs. BCCs expressed the lowest levels of CD40 and FasL. These results suggest that altered CD40 and FasL expression may be related with the progression of SCC, and the marked reduced expression of CD40 and FasL may explain the biologic behavior of BCCs.
Antigens, CD40/*metabolism ; Bowen's Disease/*metabolism ; Carcinoma, Basal Cell/*metabolism ; Carcinoma, Squamous Cell/*metabolism ; Human ; Membrane Glycoproteins/*metabolism ; Reference Values ; Skin/metabolism ; Skin Neoplasms/*metabolism

OBJECTIVETo understand the level and clinical significance of soluble CD40 in patients with chronic hepatitis B.

METHODSDetecting the concentration of sCD40 from 176 cases with chronic hepatitis B by ELISA and analyzing its relationship with different grades of inflammation and necrosis in liver tissue.

RESULTSsCD40 from patients with chronic hepatitis B was significantly higher than those from healthy. And that the concentration of sCD40 was positive correlation with severe clinical disease and liver inflammation and necrosis. In patients whose ALT lower than 80 IU/L and sCD40 higher than 80 pg/ml, it showed that 65.85% cases have high grade of liver inflammation and necrosis, which was significantly higher than patients with sCD40 lower than 80 pg/ ml.

CONCLUSIONThe concentration of sCD40 is positively related with the grade of liver inflammation and necrosis. This information could help us to evaluate the status of chronic hepatitis B as an immunological index.

Adult ; CD40 Antigens ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B, Chronic ; immunology ; metabolism ; Humans ; Male ; Solubility