OBJECTIVE: To investigate the effect of miR-125b on T cell activation in patients with aplastic anemia (AA) and its molecular mechanism. METHODS: A total of 30 AA patients were enrolled in department of hematology, Binzhou Medical University Hospital from January 2018 to October 2021, as well as 15 healthy individuals as healthy control (HC) group. Peripheral blood mononuclear cells (PBMCs) were isolated, in which the levels of miR-125b and B7-H4 mRNA were detected by RT-qPCR. Immunomagnetic beads were used to separate naive T cells and non-naive T cells from AA patients and healthy people to detect the levels of miR-125b and B7-H4 mRNA. Lentivirus LV-NC inhibitor and LV-miR-125b inhibitor were transfected into cells, and T cell activation was detected by flow cytometry. The dual-luciferase reporter gene assay was used to detect the targetting relationship between miR-125b and B7-H4. RT-qPCR and Western blot were used to detect the levels of miR-125b, CD40L, ICOS, IL-10 mRNA and B7-H4 protein. RESULTS: Compared with HC group, the expression of miR-125b was up-regulated but B7-H4 mRNA was down-regulated in PBMCs of AA patients (P <0.05), and the proportions of CD4⁺CD69⁺ T cells and CD8⁺CD69⁺ T cells in PBMCs of AA patients were higher (P <0.05). The expression of miR-125b was significantly up-regulated but B7-H4 mRNA was down-regulated in both naive T cells and non-naive T cells of AA patients (P <0.05), and non-naive T cells was more significant than naive T cells (P <0.05). Compared with NC inhibitor group, the expression of miR-125b was significantly decreased, the expression level of CD69 on CD4⁺ and CD8⁺ T cells in PBMCs was also significantly decreased, while the luciferase activity was significantly increased after co-transfection of miR-125b inhibitor and B7-H4-3'UTR-WT in the miR-125b inhibitor group (P <0.05). Compared with NC inhibitor group, the mRNA and protein levels of B7-H4 were significantly increased in the miR-125b inhibitor group (P <0.05). Compared with miR-125b inhibitor+shRNA group, the expression levels of CD69 on CD4⁺ and CD8⁺ T cells were significantly increased, and the levels of CD40L, ICOS and IL-10 mRNA were also significantly increased in the miR-125b inhibitor+sh-B7-H4 group (P <0.05). CONCLUSION MiR-125b may promote T cell activation by targetting B7-H4 in AA patients.
Humans ; Anemia, Aplastic/genetics* ; CD40 Ligand/metabolism* ; Interleukin-10 ; Leukocytes, Mononuclear/metabolism* ; Luciferases ; MicroRNAs/genetics* ; RNA, Messenger/metabolism* ; Lymphocyte Activation ; T-Lymphocytes/metabolism*

OBJECTIVE: To detect variant of the CD40L gene and infection of Jamestown Canyon virus (JCV) in a 7-year-and-9-month-old boy with co-commitment progressive multifocal leukoencephalopathy (PML) and X-linked hyper IgM syndrome (XHIGM). METHODS: Peripheral blood samples of the child and his parents were collected for the extraction of genomic DNA. The 5 exons and exon/intronic boundaries of the CD40L gene were subjected to PCR amplification and sequencing. Suspected variants were analyzed by using bioinformatic software. The JCV gene was amplified from genomic DNA by nested PCR and sequenced. RESULTS: The child was found to harbor a hemizygous c.506 A>C (p.Y169S) missense variant in exon 5 of the CD40L gene. The variant may affect the TNFH domain of the CD40L protein and result in structural instability and loss of hydrophobic interaction between CD40L and CD40. As predicted by PolyPhen2 and SIFT software, the variant was probably damaging (score = 1.00) and deleterious (score= -8.868). His mother was found to be a heterozygous carrier, while the same variant was not found in his father. Gel electrophoresis of the nested PCR product revealed presence of target JCV band, which was confirmed to be 99% identical with the JCV gene by sequencing. CONCLUSION The patient was diagnosed with co-commitment XHIGM and PML based on the testing of the CD40L gene and JCV infection.
Adult ; CD40 Ligand/genetics* ; Child ; Exons/genetics* ; Female ; Humans ; Hyper-IgM Immunodeficiency Syndrome, Type 1/genetics* ; Leukoencephalopathy, Progressive Multifocal/genetics* ; Male ; Mutation, Missense ; Polymerase Chain Reaction

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.
Animals ; Baculoviridae/genetics* ; CD40 Ligand/genetics* ; Chickens ; Cloning, Molecular ; Genetic Vectors/genetics* ; Recombinant Proteins/genetics*

A 2-year-old boy was admitted into the hospital because of cough and fever. Lymph node tuberculosis was noted when he was 2 months old and he was subsequently hospitalized several times because of cough and fever. After hospitalization the laboratory examination showed an increased eosinophia level in blood. The immune function tests shows decreased levels of IgG, IgA, and IgM. The patient had no response to anti-tuberculosis, anti-bacterial, and anti-fungal treatment, resulting in recurrent fever and progressive enlargement of the liver and spleen. Jam-like stools were noted 35 days after admission. B ultrasonography showed suspected intussusception. Laparotomy, reduction of intussusception and ileocecum angioplasty, biopsies of intestinal wall nodules and lymphoglandulae mesentericae, and hepatic biopsy were then performed under general anesthesia. The patient eventually died because of postoperative severe liver damage, disseminated intravascular coagulation and electrolyte disorder. Both the blood culture and hepatic biopsy tests showed Penicillium marneffei infecton. Immunodeficiency gene test was performed on the patient, his bother and their parents. T→G base substitution mutation (IVS1-3 T→G) in the CD40L gene was found in the patient. X-linked hyper-IgM syndrome was thus diagnosed in the patient. His mother was a carrier of the mutated CD40L gene, but his father was normal in the gene test. Hemizygous mutation in the CD40L gene was found in both the patient and his bother.
CD40 Ligand ; genetics ; Child, Preschool ; Eosinophilia ; etiology ; Fever ; etiology ; Hepatomegaly ; etiology ; Humans ; Hyper-IgM Immunodeficiency Syndrome ; diagnosis ; genetics ; Male ; Mutation ; Recurrence ; Splenomegaly ; etiology

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

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

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

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

Animals ; CD40 Ligand ; genetics ; metabolism ; Carcinoma, Hepatocellular ; genetics ; metabolism ; therapy ; Genetic Therapy ; Liver Neoplasms ; genetics ; metabolism ; therapy ; Mice ; Neoplasms, Multiple Primary ; genetics ; metabolism ; therapy ; Transgenes ; genetics

OBJECTIVETo evaluate the efficacy of cationic liposome-mediated CD40 ligand (CD40L) gene therapy for hepatocellular carcinoma.

METHODS1x10(6) of parental H22 cells or H22 cells transfected with the expression vector containing murine CD40L cDNA encoding the entire coding region (pcDNA3.1(+)-mCD40L) were inoculated subcutaneously into the left flanks of syngenic BALB/C mice. The tumor-bearing mice (tumor nodules 10 mm in maximal diameter) received the treatment of the intratumoral injection of pcDNA3.1(+)-mCD40L/Transfectam, pcDNA3.1(+), or phosphate-buffered saline (PBS), or no treatment. The mice were monitored for tumor growth weekly. We examined mCD40L messenger ribonucleic acid (mRNA) expression by reverse transcription polymerase chain reaction (RT-PCR) and the histologic changes in tumors at two weeks after intratumoral injection using immunohistochemical staining of tumor tissues.

RESULTSAll mice inoculated with parental H22 cells developed a tumor subcutaneously, and the tumor size increased progressively within three weeks. However, the mice receiving H22-CD40L cells exhibited complete regression of the tumor two weeks after tumor cell inoculation. The tumor-bearing animals with the treatment of pcDNA3.1(+) or PBS, or without treatment had progressive tumor growth, while those mice treated with pcDNA3.1(+)-mCD40L exhibited a significant inhibition of tumor growth. RT-PCR analysis showed that 783-bp fragments corresponding to the mCD40L mRNA were amplified only from pcDNA3.1(+)-mCD40L treated tumors. The tumor samples from pcDNA3.1(+)-mCD40L-treated mice showed significant lymphocyte infiltration, apoptotic bodies, and confluent necrosis in the tumor tissues.

CONCLUSIONThe tumorigenicity of CD40L-expressing cells was abrogated when the cells were implanted subcutaneously. In vivo gene therapy of established liver tumor nodules in mice by the intratumoral injection of pcDNA3.1(+)-mCD40L led to significant tumor inhibition. There was mCD40L mRNA expression in the tissues from pcDNA3.1(+)-mCD40L-treated tumors. The intratumoral injection of pcDNA3.1(+)-mCD40L induced a strong inflammatory, mainly lymphocytic infiltration of the tumor, and increased the necrotic rate of the neoplastic cells.

Animals ; CD40 Ligand ; genetics ; therapeutic use ; Carcinoma, Hepatocellular ; pathology ; therapy ; Cations ; Cell Proliferation ; Female ; Genetic Therapy ; methods ; Liposomes ; chemistry ; Liver Neoplasms ; pathology ; therapy ; Mice ; Mice, Inbred BALB C ; Transfection ; methods ; Treatment Outcome