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Objective:To discuss the effect of CD40 ligand(CD40L)on the biological behavior of the human monocytic leukemia THP-1 cells through long non-coding RNA(lncRNA)linc00239,and to clarify its potential mechanism.Methods:The linc00239 over-expression vector(pcDNA-linc00239)and interference vector(sh-linc00239)were constructed and transfected into the THP-1 cells.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the transfection efficiency.The THP-1 cells were divided into control group,vector group,pcDNA-linc00239 group,sh-linc00239 group,vector+CD40L group,pcDNA-linc00239+CD40L group,and sh-linc00239+CD40L group.RT-qPCR method was used to detect the expression levels of linc00239 in the cells in various groups;CCK-8 assay was used to detect the proliferation activities of the cells in various groups;flow cytometry was used to detect the percentages of the cells at different cell cycles and the apoptotic rates of the cells in various groups;RT-qPCR and Western blotting methods were used to to detect the expression levels of B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)mRNA and proteins in the cells in various groups;Western blotting method was used to detect the expression levels of protein kinase B(AKT)and phosphorylated AKT(p-AKT)proteins in the cells in various groups,and the ratio of p-AKT/AKT was calculated.Results:Compared with vector group,the proliferation activity of the cells and the percentage of the cells at G2 phase in pcDNA-linc00239 group were significantly increased(P<0.05 or P<0.01),the expression levels of linc00239,Bcl-2 mRNA and protein,and the ratio of p-AKT/AKT were significantly increased(P<0.05 or P<0.01),the percentage of the cells at G1 phase,apoptotic rate,and expression levels of Bax mRNA and protein in the cells were significantly decreased(P<0.05);compared with vector group,the proliferation activity of the cells and percentage of the cells at G2 phase,expression levels of linc00239,Bcl-2 mRNA and protein,and ratio of p-AKT/AKT in the cells in sh-linc00239 group and vector+CD40L group were significantly decreased(P<0.05 or P<0.01),while the percentage of the cells at G1 phase,apoptotic rate,and the expression levels of Bax mRNA and protein in the cells were significantly increased(P<0.05 or P<0.01);compared with pcDNA-linc00239 group,the proliferation activity of the cells and percentage of cells at G2 phase in pcDNA-linc00239+CD40L group were significantly decreased(P<0.05 or P<0.01),the expression levels of linc00239,Bcl-2 mRNA and protein,and ratio of p-AKT/AKT were significantly decreased(P<0.05 or P<0.01),while the percentage of cells at G1 phase,apoptotic rate,and the expression levels of Bax mRNA and protein were significantly increased(P<0.05 or P<0.01);compared with sh-linc00239 group,the proliferation activity of the cells and percentage of cells at G2 phase in sh-linc00239+CD40L group were significantly decreased(P<0.05 or P<0.01),the expression levels of linc00239,Bcl-2 mRNA and protein,and ratio of p-AKT/AKT were significantly decreased(P<0.05 or P<0.01),and the percentage of the cells at G1 phase,apoptotic rate,and expression levels of Bax mRNA and protein were significantly increased(P<0.05 or P<0.01).Conclusion:CD40L can inhibit the proliferation and cell cycle progression of the THP-1 cells through linc00239 and induce the apoptosis.
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【Objective】 To explore the effect of cilostazol on intestinal barrier function in type 2 diabetes (T2DM). 【Methods】 The GSE142153 dataset was downloaded from GEO database to analyze gene changes in diabetic patients. Eight-week-old male db/db mice and control m/m mice were randomly divided into m/m+cmc, m/m+cilo, db/db+cmc, and db/db+cilo groups. Mice in different groups were given cilostazol and corresponding solvents for 4 weeks. We detected the levels of serum sCD40L and the expression of CD40 in intestinal tissue, and evaluated the mice’s intestinal barrier function by examining intestinal permeability, water content, bacterial number, and tight junction protein expression in different groups. 【Results】 Differential expressed genes were enriched in platelet activation and endothelial barrier function pathways in diabetic patients. Compared with those in the control group, the levels of serum sCD40L in db/db diabetic mice elevated significantly, and the CD40 expression, permeability, water content and bacterial number in intestinal tissue increased obviously, while the expression of tight junction protein decreased. Cilostazol treatment in diabetic mice decreased the levels of serum sCD40L and CD40, and alleviated significantly the intestinal barrier dysfunction. 【Conclusion】 Cilostazol attenuated the damage of intestinal barrier function in T2DM, and its protective effect may be related to the inhibition of platelet activation in diabetic mice.
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Objective:To investigate the expression of soluble CD40 ligand (sCD40L) in serum of children with Kawasaki disease at acute stage and its diagnostic value in coronary artery disease (CAL).Methods:This study adopts case-control study method. Select 127 children with Kawasaki disease admitted to Xuzhou Children's Hospital affiliated to Xuzhou Medical University from August 2021 to August 2022. They are divided into CAL group and non-CAL group according to the degree of coronary artery involvement. Select 30 healthy children who have physical examination in this hospital at the same time as the healthy control group, and select another 30 children with acute upper respiratory tract infection and fever admitted to this hospital at the same time as the fever control group.Compare the sex, age and laboratory indicators of children with Kawasaki disease with or without CAL, and compare the difference between the serum sCD40L level of children with Kawasaki disease with or without CAL and the fever control group and the healthy control group, the serum sCD40L level of children with different degrees of coronary artery dilation, and analyze the correlation between the serum sCD40L and various laboratory indicators of children with Kawasaki disease and the influencing factors of children with Kawasaki disease complicated with CAL, To evaluate the screening effect of serum sCD40L for Kawasaki disease complicated with CAL. The measurement data with normal distribution is expressed by xˉ± s, the comparison between the two groups adopts independent sample t-test, the comparison between multiple groups adopts one-way ANOVA, and the comparison between two groups adopts LSD method and Bonferroni correction; The measurement data of non-normal distribution is expressed by M( Q1, Q3), and the comparison between the two groups is conducted by Mann-Whitney U test. Pearson method and Spearman mothod were used for correlation analysis. Logistic regression model was used to analyze the influencing factors of children with Kawasaki disease complicated with CAL. The diagnostic value of serum sCD40L level in Kawasaki disease complicated with CAL was analyzed by drawing the ROC curve. Results:All 127 children with Kawasaki disease were divided into CAL group (45 cases) and non-CAL group (82 cases) according to the presence or absence of CAL. The serum level of sCD40L in CAL group was higher than that in non-CAL group, healthy control group and fever control group ((7.03±0.91) μg/L vs (4.66±1.23), (1.73±0.96), (2.21±1.08) μg/L), the difference was statistically significant (all P<0.001). The serum level of sCD40L in children with coronary artery dilation in CAL group was lower than that in children with small CAA, medium CAA and large CAA ((6.04±0.22) μg/L vs (6.95±0.69), (8.02±0.57), (8.23±0.26) μg/L), the difference was statistically significant (all P<0.001). Serum sCD40L level and platelet count (PLT), C-reactive protein (CRP), N-terminal pro brain natriuretic peptide (NT-proBNP), interleukin-6 (IL-6), IL-8 and tumor necrosis factor (TNF-α) in children with Kawasaki disease All were positively correlated ( r=0.31, P<0.001, r=0.32, P<0.001, r=0.26, P=0.003, r=0.58, P<0.001, r=0.27, P=0.002, r=0.39, P<0.001). Serum sCD40L, IL-6 and NT-proBNP were the risk factors of complicated CAL in children with Kawasaki disease (odds ratio 1.21, 1.06 and 1.01, 95% confidence interval 1.03-1.43, 1.01-1.12, 1.00-1.01, P values were 0.022, 0.011 and 0.039, respectively). The area under the curve of serum sCD40L in diagnosing Kawasaki disease complicated with CAL was 0.928 (95% confidence interval: 0.885-0.971), and the optimal critical value was 5.60 μg/L, the sensitivity was 97.8% and the specificity was 79.3%. Conclusions:The level of serum sCD40L increased in children with Kawasaki disease in acute phase, especially in children with CAL. The level of serum sCD40L increased with the severity of CAL, which is a risk factor for Kawasaki disease complicated with CAL, and has certain diagnostic value for Kawasaki disease complicated with CAL.
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Pancreatic cancer is one of the most common tumors in digestive system, which is characterized by insidious clinical symptoms, strong invasion, easy metastasis and high mortality. In recent years, immunotherapy is a new direction to the treatment of solid tumors, but its applica-tion in pancreatic cancer is limited by tumor microenvironment of pancreatic cancer. The authors systematically analyze the tumor microenvironment of pancreatic cancer, summarize the clinical researches related to pancreatic cancer immunotherapy, and discuss the prospect of pancreatic cancer immunotherapy.
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CD40, a member of the tumor necrosis factor receptor (TNFR) family, is known to be involved in immune system regulation, acting as a costimulatory molecule, and in antitumor responses against cancer cells. It is a protein that is expressed in different types of cells, including immune cells and cancer cells (e.g., cervical cancer, breast cancer, melanoma). In this study, we investigated CD40/CD40L transcriptional and protein levels in cervical cancer cell lines and tumors. Higher CD40 expression was observed in cervical cancer cell lines derived from squamous cell carcinomas than from adenocarcinomas. Search of CD40/CD40L expression in cervical cancer tissues in public data sets revealed that about 83% of squamous cell carcinomas express CD40 compared to other cervical tumor subtypes. Moreover, expression of CD40 and CD40L in squamous cervical carcinomas is associated with better overall survival. Therefore, these proteins could be explored as prognostic markers in cervical cancers.
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A retrospective analysis was performed on the clinical data of a child with X-linked hyper IgM syndrome (XHIGM) with cholangiectasis as a major manifestation in Children′s Hospital of Fudan University in March 2017.The patient was a 4-year-old boy who was admitted to the hospital due to repeated diarrhea for half a year and yellow skin for 5 days.No abnormalities were found in his fetal period and birth history; The patient had 2 severe pneumonias and suppurative infection of the left axillary lymph node in infancy.Physical examination revealed delayed physical development, severe malnutrition, moderately stained yellow, lymphadenopathy and hepatomegaly.Laboratory examinations showed elevated leukocyte, eosinophils and C-reactive protein, low hemoglobin and albumin, high gamma-glutamyl transpeptidase (GGT), low IgG and normal IgM.Imaging examination revealed diffuse expansion of intrahepatic and extrahepatic bile ducts.Hepatic pathology showed hyperplasia in the bile canaliculus and some fibrous tissues around the large bile ducts.High-throughput sequencing identified a pathogenic mutation in the XHIGM gene CD 40LG (exon5 c. 506A>G, p.Y169C), with his mother as a carrier.After admission, the patient was given anti-infection, diet adjustment, albumin, intravenous immunoglobulin and ursodeoxycholic acid.The patient was discharged after the improvement in his condition.This case suggested that in addition to the common infection characteristics, XHIGM can also be manifested as diffuse intrahepatic, extrahepatic cholangiectasis and significantly elevated eosinophil.c.506A>G mutation in CD 40LG was the pathogenic mutation of this disease.
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To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.
Subject(s)
Animals , Baculoviridae/genetics , CD40 Ligand/genetics , Chickens , Cloning, Molecular , Genetic Vectors/genetics , Recombinant Proteins/geneticsABSTRACT
Objective@#To investigate the impact of macrophage-induced immune inflammation on the proliferation and apoptosis of BPH cells and its possible mechanism.@*METHODS@#Macrophages were stimulated with phorbol myristate acetate, co-cultured with BPH-1 cells, and then treated with the androgen receptor (AR) inhibitor or anti-CD40L antibody. The immunohistochemical biomarkers of the T lymphocytes (CD4 and CD8), B lymphocyte (CD20) and macrophages (CD68), AR, CD40/CD40L, and inflammatory factors IL-1, IL-6 and TNF-α were measured before and after treatment. The proliferation and apoptosis of the cells were observed by MTT assay, colony-forming assay and flow cytometry, and the expressions of cell apoptosis- and MAPK signaling pathway-related proteins were determined by qRT-PCR and Western blot.@*RESULTS@#Significantly increased proliferation and decreased apoptosis of the cells, up-regulated expressions of Bcl-2, IL-1, IL-6, TNF-α, AR, CD40 and CD40L, and down-regulated expression of Bax were observed in the BPH-1 cells co-cultured with macrophages (the M-BPH-1 group) compared with those in the blank control (B-BPH-1) group (P < 0.01). In comparison with the BPH-1 cells treated with normal saline, those treated with either low-dose CD40L (L-CD40L) or high-dose CD40L (H-CD40L) showed markedly inhibited proliferation, increased apoptosis, up-regulated expression of Bax, and down-regulated expressions of Bcl-2, IL-1, IL-6 and TNF-α (P < 0.01), and those in the low- and high-dose AR (L-AR and H-AR) inhibitor groups exhibited remarkably reduced proliferation, increased apoptosis, down-regulated expressions of Bcl-2, IL-1, IL-6 and TNF-α, and up-regulated expression of Bax (P < 0.01). The phosphorylation levels of JNK, ERK and P38 were significantly elevated in the M-BPH-1 group, but declined in the H-CD40L and the H-AR inhibitor groups compared with those in the B-BPH-1 group, all in a concentration-dependent manner (P < 0.01).@*CONCLUSIONS@#Macrophage-induced immune inflammation regulates AR and CD40/CD40L expressions and promotes the proliferation and inhibits the apoptosis of BPH-1 cells by activating the MAPK signaling pathway. /.
Subject(s)
Humans , Apoptosis , Cell Proliferation , Inflammation , Prostatic HyperplasiaABSTRACT
@#Introduction: Glycogen synthase kinase-3 (GSK-3) is an important immune regulator that controls inflammation via inhibition of its protein kinase activities. Persistent inflammatory responses through the activation of immune cells and excessive production of immune mediators may cause tissue destruction and implicated in the development of chronic inflammatory diseases. The objective of this study was to examine the role of Tideglusib, a GSK-3 inhibitor, in inflammatory responses elicited through macrophage activation by investigating the expression of cell surface biomarkers and inflammatory molecule levels. Method: The effects of GSK-3 inhibition by Tideglusib on the expression of CD11b and CD40 and secretion of pro-inflammatory cytokines in the lipopolysaccharide (LPS)-activated macrophage-derived RAW 264.7 cells were determined by flow cytometry, while the presence of nitric oxide (NO) was determined by Griess assay. Results: Stimulation of RAW 264.7 cells with LPS increased substantial levels of CD11b and CD40 expressions, and secretion of NO, TNF-α, and MCP-1. However, the expression of these molecules was suppressed through inhibition of GSK-3. Conclusion: These findings suggest the significant role of Tideglusib to limit the upregulation of immune responses in activated macrophages, and as a potential anti-inflammatory drug for the intervention and treatment of inflammatory diseases.
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Objective@#To study the effect and mechanism of the extract from grub on the CD40 of microglia cells in rabbit model of central retinal vein occlusion.@*Methods@#The 60 color rabbits were randomly divided into the blank group, model group, Xueshuantong group and sputum group. The Xueshuantong group was intragastrically administered with Xueshuantong tablets suspension 5 mg/ml, the sputum group was given gavage extract 1 g/ml, and the blank group and the model group were intragastrically administered with normal saline once daily. Except for the blank group, the other groups of rabbits were modeled by argon laser irradiation of the retinal vein trunk, and the fundus photography and FFA examination were performed immediately after modeling and at 1, 14 and 28 days after modeling, respectively, at 1, 3, 7, 14 and 28 day, the expression of CD40 in the optic nerve of rabbits was observed by immunohistochemical staining.@*Results@#The FFA results showed that the veins were not filled at 1 day after model establishment, and some veins were not developed at 14 days, and there was no evidence of revascularization at 28 days. Part of the venous in the thrombus group and the sputum group was not filled. The venous filling time was significantly shorter than that in the model group at 14 days, and the venous filling time returned to the normal level at 28 days. The results of immunohistochemistry showed that the number of microglia increased significantly at 1 d in model group. The number of microglia reached the highest peak at 3 d, and the number of microglia decreased at 7 d, 14 d and 28 d. Compared to the model group, at the 3, 7, 14, 28 d, the integrated optical density of CD40 (3 d: 8 908.91 ± 96.30, 6 099.92 ± 273.44 vs. 10 436.4 ± 1 306.8; 7 d: 5 982.06 ± 483.37, 2 957.36 ± 424.19 vs. 8 798.12 ± 444.39; 14 d: 3 225.36 ± 468.88, 342.04 ± 64.56 vs. 5 356.74 ± 439.16; 28 d: 756.97 ± 80.17, 72.85 ± 11.06 vs. 4 215.27 ± 361.00) in the Xueshuantong group and sputum group significantly decreased (P<0.05).@*Conclusions@#The thrombus and sputum extract can inhibit the activation of microglia to varying degrees, and the sputum extract is moreinhibitory effect.
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Objective To study the effect and mechanism of the extract from grub on the CD40 of microglia cells in rabbit model of central retinal vein occlusion. Methods The 60 color rabbits were randomly divided into the blank group, model group, Xueshuantong group and sputum group. The Xueshuantong group was intragastrically administered with Xueshuantong tablets suspension 5 mg/ml, the sputum group was given gavage extract 1 g/ml, and the blank group and the model group were intragastrically administered with normal saline once daily. Except for the blank group, the other groups of rabbits were modeled by argon laser irradiation of the retinal vein trunk, and the fundus photography and FFA examination were performed immediately after modeling and at 1, 14 and 28 days after modeling, respectively, at 1, 3, 7, 14 and 28 day, the expression of CD40 in the optic nerve of rabbits was observed by immunohistochemical staining. Results The FFA results showed that the veins were not filled at 1 day after model establishment, and some veins were not developed at 14 days, and there was no evidence of revascularization at 28 days. Part of the venous in the thrombus group and the sputum group was not filled. The venous filling time was significantly shorter than that in the model group at 14 days, and the venous filling time returned to the normal level at 28 days. The results of immunohistochemistry showed that the number of microglia increased significantly at 1 d in model group. The number of microglia reached the highest peak at 3 d, and the number of microglia decreased at 7 d, 14 d and 28 d. Compared to the model group, at the 3, 7, 14, 28 d, the integrated optical density of CD40 (3 d: 8 908.91 ± 96.30, 6 099.92 ± 273.44 vs. 10 436.4 ± 1 306.8; 7 d: 5 982.06 ± 483.37, 2 957.36 ± 424.19 vs. 8 798.12 ± 444.39; 14 d:3 225.36 ± 468.88, 342.04 ± 64.56 vs. 5 356.74 ± 439.16; 28 d: 756.97 ± 80.17, 72.85 ± 11.06 vs. 4 215.27 ± 361.00) in the Xueshuantong group and sputum group significantly decreased ( P<0.05). Conclusions The thrombus and sputum extract can inhibit the activation of microglia to varying degrees, and the sputum extract is moreinhibitory effect.
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The use of specific combinations of antigens and adjuvant represents a promising approach for increasing the immunogenicity of DNA vaccines. In the present study, we evaluated the immunity and antitumor effects of DNA vaccines with G250 as the target antigen in a mouse model of renal cell carcinoma. We constructed two recombinant plasmids, pVAX1-G250 and pVAX1-CD40L. The recombinant plasmids were injected into mice by intramuscular injection and electrical pulse stimulation. ELISA and ELISPOT experiments were performed to evaluate the corresponding humoral and cellular immune responses following immunization. To further investigate the antitumor potential of the DNA vaccines, we established a tumor-bearing mouse model expressing G250 target antigen. Our results showed that immunization with the combination of the two plasmids exerted the strongest anti-tumor effects. Therefore, our findings demonstrated the effectiveness of CD40L as an adjuvant for DNA vaccines and highlighted the promising use of these vaccines for the treatment of tumors.
Subject(s)
Animals , Female , Mice , DNA/classification , Vaccines/pharmacology , Immunity , Kidney Neoplasms , Carcinoma, Renal Cell/metabolism , CD40 Ligand/administration & dosageABSTRACT
There have been many recent exciting developments in biomimetic nanoparticles for biomedical applications. Inflammation, a protective response involving immune cells, blood vessels, and molecular mediators directed against harmful stimuli, is closely associated with many human diseases. As a result, biomimetic nanoparticles mimicking immune cells can help achieve molecular imaging and precise drug delivery to these inflammatory sites. This review is focused on inflammation-targeting biomimetic nanoparticles and will provide an in-depth look at the design of these nanoparticles to maximize their benefits for disease diagnosis and treatment.
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Objective :To explore influence of nicorandil on plasma levels of high sensitive C reactive protein (hsCRP) and soluble T cell CD40 ligand (sCD40L) and therapeutic effect of nicorandil in patients with microvascular angina pectoris .Methods :A total of 102 patients with microvascular angina pectoris were enrolled ,randomly and equally divided into routine treatment group (received routine comprehensive treatment ) and nicorandil group (received nic-orandil based on routine comprehensive treatment ,5mg ,3 times/d) ,both groups were treated for eight weeks . Plasma levels of hsCRP and sCD40L were measured and compared between two groups before and eight weeks after treatment ,and therapeutic effect was compared between two groups .Results : Compared with before treatment there were significant reductions in plasma levels of hsCRP and sCD 40L in both groups after eight weeks ;compared with routine treatment group after treatment ,there were significant reductions in plasma levels of hsCRP [ (2.63 ± 0.25) mg/L vs.(1.80 ± 0.28) mg/L] and sCD40L [ (71.88 ± 3.71) pg/ml vs .(55.25 ± 2.47) pg/ml] in nicorandil group , P＝0. 001 all.Total effective rate of nicorandil group was significantly higher than that of routine treatment group (78.43% vs.56.86%,P＝0.02).Conclusion :Nicorandil can significantly rise clinical effect ,reduce plasma levels of hsCRP and sCD40L in patients with microvascular angina pectoris .
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Objective:To explore the agonistic CD40 antibody(CD40ScFv and CD40mAb)-mediated suppression of mouse breast cancer cell growth and change in tumor tissue Th1/Th2 balance,as well as the mechanism underlying its antitumor effect.Methods:The re-combinant plasmid containing the CD40ScFv gene fragment was transformed into the Rosetta strain of Escherichia coli to express and purify the recombinant functional CD40ScFv protein.The 4T1 mouse breast cancer cells were cultured in vitro.Balb/C tumor model mice were divided into CD40ScFv,CD40mAb agonist,and saline(NS group)groups,which were administered CD40ScFv,CD40mAb ago-nist,and saline,respectively,to observe the change in tumor volume.The tumor tissues were removed and enzyme-linked immunosor-bent assay(ELISA)was used to determine IL-12 concentration in the tumor tissue supernatants.The tumor infiltrating lymphocytes (TILs)were extracted by enzymatic digestion.The proportion of Th1 and Th2 cells in TILs was determined by flow cytometry.Results:The CD40ScFv protein was successfully identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot.The molecular size and concentration of the purified protein were 27kDa and 1.12 mg/mL,respectively.The tumor sizes of the CD40ScFv and CD40mAb groups were(3.044±0.239)cm3and(2.749±0.261)cm3,respectively,which were significantly smaller(P<0.05)than that of the NS group(3.933±0.326)cm3.The tumor IL-12 concentration(determined by ELISA)in the CD40ScFv group(396.27±48.13)pg/mL and the CD40mAb agonist group(457.63±58.37)pg/mL were significantly higher(P<0.05)than that of the NS group(79.51±14.97) pg/mL.The results of flow cytometry showed that the excited Th1/Th2 cell ratios were 6.32±0.87 and 5.54±0.71 for the CD40ScFv and CD40mAb groups,respectively,which were significantly higher(P<0.05)than that of the NS group(1.79±0.38).Conclusions:The agonis-tic CD40 antibody inhibited tumor growth by regulating the Th1/Th2 ratio and IL-12 secretion via promotion of DC activation,which is one of the important mechanisms affecting Th1/Th2 balance in the tumor microenvironment
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Objective To investigate whether follicular helper T(Tfh) cells were involved in the development of Henoch-Sch?nlein purpura(HSP) and Henoch-Sch?nlein purpura nephritis(HSPN) in chil-dren through affecting CD40/CD40L axis. Methods Fifty-five subjects were enrolled in this study and di-vided into four groups as follows:22 children with HSP but without renal involvement(Group A),11 chil-dren with HSPN presenting with microhematuria(Group B),11 children with HSPN presenting with micro-hematuria and proteinuria (Group C) and 11 healthy children (control group). Flow cytometry was per-formed to detect the percentages of CD19+B cells and their subsets,CD19+B cells and CD19+CD38+B cells secreting different Ig classes,CD19+CD40+B cells and their subsets and Tfh cells expressing CD40 ligand (CD40L). Results Compared with the control group,the percentages of CD19+CD86+B,CD19+CD138+B and CD40L+Tfh cells significantly increased in Group C(P<0.05) and slightly increased in Groups A and B (P>0.05). No significant difference in the percentages of CD19+B cells, CD19+CD27+B cells, CD19+B cells or CD19+CD38+B cells expressing IgG, IgM, IgD, CD19+B cells or CD19+B cell subsets secreting CD40 was found between the control group and Groups A,B and C(P>0.05). Moreover,the percentages of CD19+B and CD19+CD38+B cells secreting IgA and IgE in Groups A,B and C were higher than those in the control group(P<0.05). Secretion of IgA by CD19+B and CD19+CD38+B cells were positively correla-ted with the expression of CD40L by Tfh cells(P<0.05). Conclusion Tfh cell-mediated abnormal expres-sion of CD40/CD40L might play an important role in the development of HSP and be related to the clinical severity of renal involvement in HSPN.
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Objective To obtain a mouse CD40-specific single-chain antibody (scFv) with high purity and to investigate its in vivo and in vitro agonistic activity on natural killer(NK) cells against tumor. Methods Agonistic anti-mouse CD40 scFv with high purity was obtained by genetic engineering. Mouse dendritic cells (DCs) were treated with different strategies including anti-CD40 monoclonal antibody (McAb),anti-CD40 scFv and negative control. Expression of IL-12 in the supernatants of DC cultures was measured by ELISA. Then DCs and NK cells were co-cultured to obtain activated NK cells,which were co-cultured with T6-17 cells. WST-8 was used to test the cytotoxic effects of NK cells on T6-17 cells. A mouse tumor model was established by injecting BALB/c nude mice with T6-17 cells. Anti-CD40 scFv was injected into tumor to evaluate its inhibitory effect on tumor growth. Levels of IL-12 and IFN-γ in the tumor microen-vironment were detected by ELISA. Changes in the percentages of tumor-infiltrating NK cells and the expres-sion of NKG2D protein(natural-killer group 2,member D) were detected by flow cytometry and immunohis-tochemistry,respectively. Results The recombinant plasmid CD40 scFv-pET28a was confirmed to be con-structed correctly. Results of SDS-page and His-tag Western blot revealed that anti-CD40 scFv could be ex-pressed successfully with a relative molecular mass of 27×103. The level of IL-12 in the supernatant of DC culture of anti-CD40 scFv group was (555.86 ±40.48) pg/ml, which was significantly higher than that of negative control group (P<0.05). The killing ability of NK cells in anti-CD40 scFv group was (72.23 ± 3.99)%,which was significantly higher than that in negative control group (P<0.05). Anti-CD40 scFv significantly inhibited the tumor growth in BALB/c nude mice as compared with negative control group(P<0.05). The levels of IL-12 and IFN-γ in tumor microenvironment of anti-CD40 scFv group were(188.801± 32.718) pg/ml and(121.428±30.994) pg/ml,respectively,which were significantly higher than those in normal saline(NS) group(P<0.05). The positive rate of NKG2D protein and the percentage of CD3-DX5+cells in anti-CD40 scFv group were respectively(8.18±2.01)% and(19.15±2.24)%,which were signifi-cantly higher than those in NS group (P<0.05). Conclusion Agonistic anti-mouse CD40 scFv could en-hance the anti-tumor ability of NK cells by activating DCs.
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X-linked immunodeficiency with hyper-immunoglobulin(Ig) M is a primary immunodeficiency disease,mainly due to the mutation of coding CD40 ligand(CD40L),which results in the dysfunction of immunoglobulin class switching that causes in a normal or increased IgM level,and a marked decrease in IgG,IgA and IgE level.The clinical manifestations are recurrent infection,neutropenia,and autoimmune disease or tumor.
ABSTRACT
Abstract Cluster of differentiation 40 (CD40), the receptor for CD154, is a member of the tumor necrosis factor (TNF) receptor superfamily. Several studies have been conducted to investigate the effect of the CD40 rs1883832 polymorphism on atherosclerotic disease in different population; however, inconsistent results were obtained. In this study, we investigated the association of four polymorphisms (rs1883832, rs13040307, rs752118 and rs3765459) of CD40 gene and their effect on CD40 expression with the risk of ischemic stroke (IS) in a Chinese population. Three hundred and eighty patients with IS and 450 control subjects were included in the study. The CD40 polymorphisms were discriminated by Snapshot SNP genotyping assay. Serum soluble CD40 (sCD40) levels were detected by ELISA. We found that the rs1883832CT and rs1883832TT genotypes were associated with an increased risk of IS compared with the rs1883832CC genotype (OR = 1.42, 95% CI: 1.03-1.95, p = 0.030 and OR = 1.91, 95% CI: 1.29-2.82, P = 0.001, respectively), and the rs1883832T allele was associated with a significantly increased risk of IS compared with rs1883832C allele (OR = 1.40, 95% CI: 1.15-1.70, P = 0.001). Elevated serum sCD40 levels were observed in patients with IS compared with the control gropu (P < 0.01). Individuals carrying the rs1883832TT or rs1883832CT genotypes showed significantly higher sCD40 levels compared with the rs1883832CC genotype in the IS group [(64.8 ± 25.4 pg/mL, TT = 94); (63.9 ± 24.3 pg/mL, CT = 185) vs (53.3 ± 22.5 pg/mL, CC = 101), P < 0.01]. The TCCA haplotype was associated with an increased risk of IS compared with the control group (OR = 2.10, 95% CI: 1.23-3.58, p = 0.005). However, we did not find a significant association between the other three polymorphisms and IS risk. In conclusion, after a comprehensive comparison with other studies, we confirmed that the rs1883832T allele but not the rs1883832C allele is associated with an increased risk of IS. The rs1883832 polymorphism may exert influences on abnormal CD40 expression in IS patients among the Chinese population.
ABSTRACT
Focal segmental glomerulosclerosis (FSGS) in children, which is a kind of nephrotic syndrome showing steroid resistance, usually progresses to a substantial number of end stage renal disease (ESRD). Although the pathogenesis of primary FSGS is unclear, several recent studies have reported that FSGS is associated with circulating immune factors such as soluble urokinase-type plasminogen activator receptor (suPAR) or anti-CD40 autoantibody. We report a successfully treated case of a 19-year-old female patient who experienced a recurrence of primary FSGS. After the diagnosis of FSGS, the patient progressed to ESRD and received a kidney transplantation (KT). Three days later, recurrence was suspected through proteinuria and hypoalbuminemia. She has been performed plasmapheresis and high dose methylprednisolone pulse therapy and shown remission status without increasing proteinuria for four years after KT. In conclusion, strong immunosuppressive therapy may be helpful for a good prognosis of recurrent FSGS, suppressing several immunologic circulating factors related disease pathogenesis.