共信号分子CD40/CD40L质粒标准品的构建

背景:目前,对CD40/D40L的研究主要集中在可溶性和蛋白方面,对其在转录水平上的研究文献不多,可能与其无商品化质粒标准品有关.目的:构建检测CD40/CD40L mRNA表达水平差异的标准品.设计、时间及地点:单一样本实验,于2009-04在苏州大学附属第一医院检验科实验室完成.材料:取手术切除的子宫肌瘤组织,经患者知情同意.方法:以手术切除的子宫肌瘤组织细胞的总RNA为模板、Random 6mers为引物反转录合成cDNA,用该cDNA为模板PCR扩增人CD40/CD40L基因相应的cDNA片断,构建PMD-18-CD40/CD40L重组质粒,鉴定测序后,用荧光定量PCR制作标准曲线.主要观察指标:①RNA质量检测结果.②扩增的目的片段电泳结果.③CD40/CD40L基因测序结果.④标准品的扩增情况.结果:组织提取的总RNA完整性良好,构建的CD40/CD40L质粒经PCR扩增后分别得到136 bp,200 bp的清晰条带,测序结果与目的片段完全一致,且质粒的原始浓度为2.66×10~(13)、2.45×10~(13) copies/mL,倍比稀释至2.0×10~6 copies/mL均能得到良好的标准曲线(R~2=1).结论:实验成功构建了CD40/CD40L基因荧光定量PCR标准质粒.     

恶性淋巴瘤血清中可溶性CD40配体与肿瘤坏死因子α表达的临床意义

目的研究恶性淋巴瘤（ML）患者血清中可溶性CD40配体（sCD40L）和肿瘤坏死因子α（TNF-α）的表达，分析血清中sCD40L和TNF-α的关系。方法应用酶联免疫吸附法检测39例ML（其中初治17例，复发病例22例）血清中sCD40L和TNF-α水平；同时检测正常人20例血清中sCD40L和TNF-α水平。结果ML患者血清sCD40L表达明显低于对照组（P〈0．01）；TNF-α明显高于对照组（P〈0．01）。高度恶性ML血清sCD40L水平（0．06±0．03）μg／L比中、低度恶性（0．38±0．13）μg／L，（0．82±0．26）μg／L明显降低（P〈0．01）。高度恶性ML血清TNF-α水平（50．74±6．63）ng／L比中、低度恶性（34．29±8．34）ng／L、（23．33±6．64）ng／L明显升高（P〈0．01）。化疗缓解组血清sCD40L水平（0．96±0．20）μg／L比无效组（0．28±0．236）μg／L明显升高，TNF-α水平缓解组（13．74±9．02）ng／L，无效组（37．23±10．73）ng／L明显降低（P〈0．01）。ML患者血清sCD40L水平和TNF-α水平呈负相关（r=-0．233，P〈0．05）。结论恶性淋巴瘤患者血清sCD40L表达降低，TNF-α表达升高；二者在血清中的表达有一定的相关性。sCD40L有可能作为预测恶性淋巴瘤预后的一个指标。     

Immune alterations in malignant melanoma and current immunotherapy concepts

Introduction: Malignant melanoma is a highly aggressive, immunogenic tumor that has the ability to modulate the immune system to its own advantage. Patients with melanoma present numerous cellular immune defects and cytokine abnormalities, all leading to suppression of the host anti-tumor immune response. Innovative treatment strategies can be achieved through employing our knowledge of the melanoma-induced immune alterations.

Areas covered: The authors review comprehensively the immune abnormalities in individuals with melanoma, and provide a summary of currently available melanoma immunotherapy agents that are currently on the market or undergoing clinical trials.

Expert opinion: Ipilimumab, a monoclonal antibody directed against the CTLA-4, is one of the current forefront treatment strategies in malignant melanoma. Novel immunomodulating agents have shown clear activity in patients with malignant melanoma. These include anti-PD-1 and anti-PD-1 ligand antibodies that may soon become important items in the anti-melanoma armamentarium. Combinations of different immunotherapy agents, between themselves or with other agents, are currently being studied in an attempt to further enhance the antineoplastic effect in patients with malignant melanoma.     

Glucocorticoids upregulate CD40 ligand expression and induce CD40L-dependent immunoglobulin isotype switching

IL-4 and CD40 ligation are essential for IgE synthesis by B cells. We have shown previously that hydrocortisone (HC) induces IgE synthesis in IL-4-stimulated human B cells. In this study we demonstrate that HC fails to induce IgE synthesis in B cells from CD40 ligand-deficient (CD40L-deficient) patients. Disruption of CD40L-CD40 interactions by soluble CD40-Ig fusion protein or anti-CD40L mAb blocked the capacity of HC to induce IgE synthesis in normal B cells. HC upregulated CD40L mRNA expression in PBMCs and surface expression of CD40L in PBMCs as well as in purified populations of T and B cells. Upregulation of CD40L mRNA in PBMCs occurred 3 hours after stimulation with HC and was inhibited by actinomycin D. Upregulation of CD40L mRNA and induction of IgE synthesis by HC were inhibited by the steroid hormone receptor antagonist RU-486. These results indicate that ligand-mediated activation of the glucocorticoid receptor upregulates CD40L expression in human lymphocytes.     

Soluble CD40 ligand can replace the normal T cell-derived CD40 ligand signal to B cells in T cell-dependent activation

We have constructed a soluble chimeric fusion protein between the mouse CD8 alpha chain and the mouse CD40 T cell ligand. This protein binds to both human and mouse B cells. By itself it induced a modest degree of B cell proliferation, but together with anti-immunoglobulin (anti-Ig) antibody it greatly stimulated B cell proliferation, as determined by both [3H]thymidine uptake and increase in cell numbers. These data are evidence that the CD40 ligand on T cells provides a signal that drives B cell proliferation. This signal is synergistic with that delivered by anti-Ig antibody.     

Upregulation of CD40--CD40 ligand system in patients with diabetes mellitus

BACKGROUND: Diabetes is associated with an increased risk of cardiovascular disease and atherosclerosis. Increasing evidence shows that CD40-CD40L interaction plays a crucial role in the pathogenesis of atherosclerosis and coronary artery disease. The purpose of this study was to assess whether CD40 system expressions were disrupted in patients with diabetes. METHODS: Sixteen normal controls and 72 patients including 20 with type 2 diabetes mellitus (DM), 15 with type 1 DM, 20 with coronary heart disease (CHD) and 17 CHD with coexisting DM were investigated. The expression of CD40 and CD40L on platelet was analyzed by indirect-immunofluorescence flow cytometry and serum-soluble CD40L level was determined by a commercially available ELISA. Serum of AGE was detected by fluorescence spectroscopy. RESULTS: Type 1 DM, type 2 DM, CHD and CHD Patients with coexisting diabetes showed a significant increase of CD40 (81.8 +/- 11.7, 70.7 +/- 11.6, 68.5 +/- 10.2, 79.9 +/- 11.9 MIF, respectively) and CD40L (18.4 +/- 5.1, 13.9 +/- 4.1, 13.5 +/- 3.7, 16.7 +/- 4.7 MIF, respectively) coexpression on platelets as well as sCD40L (15.6 +/- 3.5, 14.1 +/- 3.3, 12.2 +/- 3.5, 13.5 +/- 3.6 ng/ml, respectively) compared with controls (p < 0.01). A positive correlation was found between serum AGE levels in patients with DM and CD40-CD40L system. We also observed a significant correlation between hemoglobinA1c (HbA1c) concentration and CD40L on platelets (r = 0.71, p < 0.001) as well as sCD40L (r = 0.69, p < 0.001), but not for CD40 on platelets. CONCLUSIONS: Patients with diabetes show increased coexpression of CD40 system, especially CD40L, which may create a proinflammatory and prothrombotic milieu for aggravating the development of atherosclerosis.     

Increased soluble CD40 ligand levels in cystic fibrosis

Summary. Chronic inflammation represents a key pathogeneric event in the progression of lung disease in cystic fibrosis (CF). To identify novel mechanisms of the inflammatory reaction in CF and analyze its relation with coagulative activation, we carried‐out a cross‐sectional study to evaluate circulating levels of the inflammatory mediators soluble (s) CD40L, C‐reactive protein (CRP), interleukin (IL)‐1β, the coagulation markers activated factor VII (FVIIa) and prothrombin fragment (F) 1+2, as well as urinary 11‐dehydro‐thromboxane (TX)B₂, an index of in vivo platelet activation, in 34 CF patients and 34 matched healthy subjects. We observed that CF patients displayed significantly increased circulating levels of sCD40L compared to controls [2.8 (0.4–15.6) vs 1.1 (0.2–2.7) ng mL^?1 ,P = 0.0003]. sCD40L levels inversely correlated with forced expiratory volume at 1 second (FEV₁) (ρ = ?0.788, P = 0.0001), whereas it directly correlated with CRP and IL‐1β levels (ρ = 0.621, P = 0.0004; and ρ = 0.745, P = 0.0001, respectively), which were also elevated in CF patients. CF patients had also enhanced levels of FVIIa and F1+2 compared to controls [39.2 (22.6–69.8) vs 22.3 (16.2–32.4) mU mL^?1, P = 0.0001; 0.60 (0.30–1.80) vs 0.17 (0.10–0.40) nmol L^?1, P = 0.0001, respectively]. A direct correlation was observed between sCD40L and both plasma FVIIa (ρ = 0.691, P = 0.0001) and F1+2 (ρ = 0.545, P = 0.0017) as well as between sCD40L and urinary 11‐dehydro‐TXB₂ (ρ = 0.433, P = 0.0129). Our findings suggest that in CF patients, sCD40L could represent a biochemical link between the inflammatory state, and endothelial damage and coagulative activation, leading to progressive impairment of pulmonary function.     

Increased levels of CD40-CD40 ligand system in patients with essential hypertension

BACKGROUND: Although it has been hypothesized that hypertension is in part an inflammatory disorder, clinical data linking inflammation with incident hypertension are scarce. There is evidence that have shown that CD40-CD40L interaction plays a pathogenic role in inflammatory disorders. We assessed whether CD40 system expressions were altered in patients including 30 with hypertension grade 1, 80 with hypertension grade 2 and 40 with hypertension grade 3. METHODS: Twenty normal controls and 150 patients with essential hypertension were investigated. The expression of CD40 and CD40L on platelet was analyzed by indirect-immunofluorescence flow cytometry and soluble CD40L level was determined by a commercially available ELISA. C-reactive protein was also measured by ELISA. RESULTS: All patients with hypertension showed a significant increase of CD40 (67.1+/-9.6 Mean Fluorescence Intensity, MFI) and CD40L (15.3+/-5.0 MFI) coexpression on platelets as well as sCD40L levels (12.8+/-3.9 ng/ml ) compared with controls (p<0.0001). We found that CRP levels related to CD40-CD40L system. We also observed a slight correlation between sCD40L level and blood pressure. During 3 months follow-up, patients with enhanced levels of sCD40L (>15 ng/ml) indicated a tough control of blood pressure. CONCLUSION: Patients with essential hypertension show increased coexpression of CD40 system, which suggests that hypertension is in part an inflammatory disorder.     

Melanoma immunotherapy by targeted IL-2 depends on CD4(+) T-cell help mediated by CD40/CD40L interaction

The induction of tumor-protective immunity against malignancies remains a major challenge in cancer immunotherapy. A novel, humanized anti-ganglioside-GD(2)-IL-2 immunocytokine (hu14.18-IL-2) induced CD8(+) T cells to eradicate established pulmonary metastases of B78-D14 murine melanoma, in a process that required help by CD4(+) T cells and was mediated by the CD40/CD40 ligand (CD40L) interaction. The anti-tumor effect was diminished in mice deficient in CD4(+) T-cells. Three lines of evidence show that CD4(+) T-cell help was mediated by CD40/CD40L interaction but not by endogenous IL-2 production. First, the hu14.18-IL-2-induced anti-tumor response is partially abrogated in C57BL/6J CD40L knockout (KO) mice in contrast to C57BL/6J IL-2 KO animals, in which the immunocytokine was completely effective. Second, partial abrogation of the anti-tumor effect is induced with anti-CD40L antibodies to the same extent as with CD4(+) T-cell depletion. Third, a complete anti-tumor response induced by hu14.18-IL-2 can be reconstituted in C57BL/6J CD40L KO mice by simultaneous stimulation with an anti-CD40 mAb. These results suggest that help provided by CD4(+) T cells via CD40/CD40L interactions in our tumor model is crucial for effective immunotherapy with an IL-2 immunocytokine.     

BCG immunotherapy as a systemic adjunct to surgery in malignant melanoma.

Results of our study suggest that BCG systemic adjuvant immunotherapy may be effective for improving both the recurrence and survival rates in patients with regional metastases from malignant melanoma. BCG is more effective in patients with small amounts of subclinical disease. It is apparent from results of clinical trials that many of the principles derived from the study of animal tumor systems are applicable to human cancer in that immunotherapy is most effective for a small residual number of tumor cells. BCG treatment fulfills many of the ideal criteria for adjuvant treatment following surgery. It is relatively nontoxic; it is effective for disseminated melanoma; it has systemic activity in the adjuvant treatment of subclinical metastases. Hwever, until clinical trials are complete, this treatment as adjuvant therapy must be considered investigational.     

Cyclosporin A inhibits CD40 ligand expression in T lymphocytes.

The ligand for CD40 is expressed on activated T lymphocytes and delivers contact-dependent activation signals to B lymphocytes. The mechanisms regulating CD40 ligand gene expression are largely unknown. Optimal expression of CD40 ligand required activation of protein kinase C and a rise in intracellular calcium concentration. CD40 ligand expression was inhibited by pretreatment of T cells with cyclosporin A. Cyclosporin A analogues inhibited CD40 ligand expression with a potency mirroring the ability of each compound to inhibit calcineurin activity, indicating that calcineurin plays a key role in CD40 ligand gene expression. Cyclosporin A inhibited IL-4-driven CD40 ligand-dependent IgE isotype switching in PBMC but did not inhibit IgE synthesis induced by CD40 mAb plus IL-4. PBMC derived from transplant patients receiving cyclosporin A failed to express CD40 ligand upon stimulation. These results suggest that patients receiving cyclosporin A may be deficient in CD40 ligand-dependent T cell help.     

CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligand for CD40

CD40 is a member of the tumor necrosis factor (TNF) receptor family of cell surface proteins and was originally described as a B cell restricted antigen. Treatment of primary human monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), or interferon gamma (IFN-gamma) resulted in the induction of CD40 mRNA and enhancement of cell surface protein expression. CD40 was found to mediate monocyte adhesion to cells expressing recombinant CD40 ligand. CD40 ligand-transfected cells provided a potent costimulus for monocyte TNF-alpha and IL-6 production in the presence of GM-CSF, IL-3, or IFN-gamma, and enhanced IL-8 production stimulated by GM-CSF or IL- 3. In addition, CD40 ligand-transfected cells acting in the absence of a costimulus induced monocytes to become tumoricidal against a human melanoma cell target. Collectively, these data indicate that CD40 ligand is pleiotropic with potent biological activity on monocytes.     

糖皮质激素对多发性硬化患者外周血淋巴细胞CD40和CD40L表达的影响

目的 探讨多发性硬化(MS)患者外周血淋巴细胞上CD40和CD40配体(CD40L)的表达和糖皮质激素(GC)治疗对CD40和CD40L表达的影响.方法 利用流式细胞仪对MS急性发作患者(Ms组,32例)GC治疗前后及健康对照组(30例)外周血淋巴细胞中CD40和CD40L进行检测.结果 治疗前MS组淋巴细胞中CD40和CD40L的表达较健康对照组显著升高,分别为CD40(29.67±9.95)%vs(26.60±6.28)%(P＜0.05)和CD40L(9.65±3.62%)VS(6.90±1.66)%(P＜0.01);GC治疗后淋巴细胞CD40L表达较治疗前显著降低(8.27±2.28)%vs(10.07±4.00)%(P＜0.05),而CD40表达较治疗前显著增高(34.75±7.86)%VS(29.51±9.95)%(P＜0.05).结论 Ms急性发作时患者外周血淋巴细胞上CD40和CD40L的表迭明显增高,GC对MS患者淋巴细胞CD40L表达有调节作用,对细胞免疫有抑制作用.     

Soluble CD40 ligand in systemic lupus erythematosus and antiphospholipid syndrome

AIM: To investigate a clinical role of soluble (s) CD40 ligand in systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). MATERIAL AND METHODS: A serum concentration of sCD40 ligand was measured with enzyme immunoassay (Bender Medsystems, Austria) in 21 patients with primary antiphospholipid syndrome (PAPS), in 25 patients with secondary APS (SAPS) associated with SLE, in 92 SLE patients and in 16 healthy donors. RESULTS: A sCD40 ligand concentration in sera of SAPS and SLE patients was significantly higher than in donors. Significant differences by the ligand level between the above patients were not seen. In PAPS sCD40 ligand concentration was normal. Elevated serum concentration of the ligand was observed in 9.5% patients with PAPS, 54.0%--with SAPS, 73.9%--with SLE. This rise in SLE and SAPS was not related with the disease activity or renal damage. Hyperexpression of the ligand in APS was associated neither with thromboses nor with a high concentration of IgG/IgM antibodies to cardiolipin. A direct correlation occurred between sCD40 ligand level and platelet count. In SLE and SAPS elevation of the ligand level correlated with increased thickness of carotid artery intima-media complex, hypercholesterinemia and diastolic dysfunction of left ventricular myocardium. CONCLUSION: Hyperexpression of sCD40L in SLE and SAPS is associated with developing cardiovascular diseases and atherosclerosis.     

Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes.

The specificity of T cell help for B cell activation and differentiation is maintained by the brief expression on the T cell surface, following T cell receptor-mediated triggering, of CD40 ligand (CD40L). Interaction of T helper (Th) cell CD40L with B cell CD40 induces B cell activation, cell surface expression of activation antigens, proliferation, and initiation of immunoglobulin isotype switch. We predicted that in patients with systemic lupus erythematosus (SLE), in whom Th cell-dependent production of autoantibodies results in immune complex-mediated tissue damage, CD40L expression might be augmented, prolonged, or abnormally regulated. Baseline expression of CD40L was increased in some SLE patients studied, when compared with control subjects. While Th cells from normal subjects (n = 14) and rheumatic disease control patients (n = 9) showed maximal expression of CD40L, after in vitro activation with phorbol myristate acetate (PMA) and ionomycin, at 6 h of culture with diminished levels observed at 24 and 48 h, Th cells from SLE patients (n = 19) maintained high level cell surface expression of CD40L through 24 and 48 h of culture. The prolonged expression of CD40L was functionally significant, as 24 h-activated SLE T cells, when cocultured with target B cells, induced greater B cell surface CD80 (B7-1) expression than did 24 h-activated normal T cells. These results document impaired regulation of CD40L expression in SLE T cells and identify an important potential target for therapy in this systemic autoimmune disease.     

Disruption of antigen-induced inflammatory responses in CD40 ligand knockout mice.

The objective of this study was to investigate the contribution of the interaction between CD40 and its ligand (CD40L) to antigen-induced airways inflammatory responses. To this end, we used a model involving ovalbumin (OVA) sensitization followed by OVA aerosol challenge in CD40L knockout (KO) mice. OVA-specific IgE and IgG1 were detected in the serum of the sensitized control, but not in CD40L-KO mice. After antigen challenge, sensitized control mice developed airway inflammation that was primarily eosinophilic. This inflammatory response was dramatically reduced in CD40L-KO mice. In contrast, similar numbers of eosinophils were observed in both the bone marrow and the peripheral blood in the sensitized controls and mutant strains after antigen challenge. To investigate the mechanisms underlying these findings, we examined levels of the cytokines IL-5, IL-4, and TNFalpha in both bronchoalveolar lavage (BAL) and serum. Similar levels of IL-5 were detected in BAL and serum of control and CD40L-KO mice; however, negligible levels of IL-4 in BAL and serum and of TNFalpha in BAL were detected in CD40L-KO mice when compared with control mice. Furthermore, we demonstrated that endothelial cell expression of vascular cell adhesion molecule 1 in OVA-sensitized and -challenged CD40L-KO mice was, as detected by immunohistochemistry, markedly decreased compared with that observed in similarly treated control mice. In addition, we locally overexpressed IL-4 and TNFalpha by using an adenoviral (Ad)-mediated gene transfer approach. Intranasal administration of either Ad/TNFalpha or Ad/IL-4 into OVA-sensitized and -challenged CD40L-KO mice did not reconstitute airway eosinophilia. However, concurrent administration of Ad/TNFalpha and Ad/IL-4 upregulated endothelial expression of vascular cell adhesion molecule 1, and resulted in full reconstitution of the inflammatory response in the airways. Together, these findings demonstrate the importance of the CD40-CD40L costimulatory pathway in the full expression of the inflammatory response in the airways.