Gene cloning, protein expression and examination of biological activity of chicken CD40L / 生物工程学报

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.

Genetic analysis of a child with co-commitment progressive multifocal leukoencephalopathy and X-linked hyper IgM syndrome / 中华医学遗传学杂志

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.

Designing of relative quantitative Real-Time PCR for detection of CD40 gene expression in dendritic cell after suppression with antisense

Dendritic cells have a critical role in control and regulation of immune responses. It is believed that these cells can be used for the treatment of many diseases. One of the methods used in immunotherapy is based on generating of tolerogenic dendritic cells through inhibition of expression costimulatory molecules. CD40 is one of the costimulatory molecules, and inhibition of expression by antisense or siRNA techniques, can generate tolerogenic dendritic cells. Generation of tolerogenic dendritic cells will be useful in the treatment of many diseases. By developing a quantitive RT-PCR for evaluation of gene expression, generation of these cells could be possible. Using proper software we designed an Antisense and transfection of dendritic cells by lipofectamine 2000 [Invitrogen] could lead us to generate tolerogenic dendritic cells. In this study dendritic cells were extracted from of Balb/c mice Spleen and the purity of this extraction was determined by flow cytometry. BCL 1 cell line as a CD40 expressing control group and Wehi-164 cell line were cultured in RPMI-1640+10%FCS. Primer design for CD40 gene and house keeping gene [GADPH] was done by bioinformatic soft wares such as Beacon designer, mfold and Blast. RNasy plus mini kit [Qiagen] was used for RNA extraction and the Purity and Integrity were determined by O.D at 260/280 and agarose gel electrophoresis. In the next step cDNA synthesized and quantitative RT-PCR for CD 40 using IQ sybergreen [Biorad] were setup. Finally, standard curve for CD40 and internal control in different RNA concentrations were performed. After transfection with lipofectamin 2000 the amount of gene suppression were quantified by qualitative RT-PCR. Using gradient real time PCR, optimum annealing temperature, C[t] and delta Rn for CD40 and GADPH were determined, annealing temperature was 59.5 degree sign c and melting temperature was 84 degree sign c. Slope of the curve and the efficacy of PCR for CD40 and GADPH genes were quantified by serial dilution method. CD 40 Standard curve efficiency is 96.5 and the slope is -3.408 and the efficacy of GADPH standard curve is 94.1 and its slope is -3.471. The amount of CD40 gene suppression by antisense in dendritic cells was 1/32 and in BCL1 cell line was 1/64. Also the transfection reagent had no effect on the gene expression. The best time for CD40 gene expression is 48h after transfection. Semi quantitative PCR, absolute quantitative PCR and relative quantitative PCR are used to evaluative the expression of different genes such as CD40. In this study we found that for making CD40 and GADPH standard curves, Biorad two steps PCR kit [IQ -sybergreen] and Oligo dt for cDNA synthesis were very suitable. In this case, GADPH is a good internal control. Also for evaluation of gene suppression relative quantitative RT-PCR was more efficient compare to other methods such as northern blotting. The CD40 gene Suppression after 48 hours in dendritic cells was 1/32 and in BCL1 cells was 1/64

De novo mutation causing X-linked hyper-IgM syndrome: a family study in Taiwan.

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency disorder caused by mutations of the gene encoding the CD40 ligand (CD40L). It is characterized by recurrent infections with markedly decreased serum IgG, IgA and IgE levels but normal or elevated IgM levels. We report the clinical manifestations and complete immune studies in the first family with molecularly proven XHIM in Taiwan. A 5-month-old boy presented with rapidly progressive pneumonia which responded poorly to antibiotics. High levels of IgM and very low levels of IgG, IgA, and IgE were noted in his plasma specimen: IgM, 128 mg/dl; IgG, 18 mg/dl; IgA, 4 mg/dl); IgE, 1 IU/ml. Whole blood flow cytometry when he was 21 months old showed that only a small percentage (0.48%) of his in vitro-activated CD4+ T cells expressed CD40L. When he was 3 years old, repeated flow cytometry showed essentially the same result (0.4%), compared with his father's CD40L expression of over 85%. The patient's mother had moderately decreased CD40L expression (74.4%). Hyper-IgM syndrome was confirmed by CD40L mutation analysis in the boy, which revealed a Lys 96 stop (nucleotide A307T) in exon 2 of CD40L, with a truncated protein resulting in the loss of the entire TNF domain. His mother was a carrier and apparently the individual in whom the mutation originated. Eleven other family members, including the patient's father, sister, and grandmother, and the mother's sisters and their children, all had normal results on CD40L mutation analysis. The patient has remained without significant bacterial infection on a regimen of monthly IVIG infusion and oral trimethoprim-sulfamethoxazole for Pneumocystis carinii pneumonia (PCP) prophylaxis, although he has had recurrent oral ulcers and neutropenia. Bone marrow transplantation is planned.

Activation of B cells using Schneider 2 cells expressing CD40 ligand for the enhancement of antigen presentation in vitro

CD40 ligand (CD40L) expressed by activated CD4+ T cells is a family member of membrane bound TNF family ligand and its interaction with CD40 expressed in APC has been shown to contribute in enhancing immune response. Exogenous stimulation through CD40 has been performed using soluble trimeric CD40L, anti-CD40 monoclonal antibody and cells expressing CD40L. Schneider 2 (S2) cells, a cell line derived from Drosophila melanogaster, was transfected with a plasmid vector, pAc5.1/V5-HisA, for the constitutive expression of CD40L (S2-CD40L). Upon incubation of S2-CD40L with B-lymphocytes for 6 days, activated B cells were examined by counting B cell numbers and for activation markers including CD86 and HLA Class II molecules. The activated B cells were tested for its efficient APC function by mixed lymphocyte reactions (MLR) and enzyme-linked Immunospot (ELISPOT) assay. S2-CD40L was cultured for a year and maintained CD40L expression (>90%). S2-CD40L induced B cell activation as demonstrated by increment of total B cells and up-regulation of CD86 and MHC Class II molecules. Activated B cells pulsed with peptide from human cytomegalovirus pp65 antigen efficiently induced both proliferation and IFN-gamma secretion of T cells. Our result suggests that S2-CD40L can efficiently and conveniently generate B cells as a functional APC and represents a potential role for B-cell mediated cancer immunotherapy.

X-linked Hyper-IgM Syndrome Associated with Cryptosporidium parvum and Cryptococcus neoformans Infections: the First Case with Molecular Diagnosis in Korea

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency disorder, caused by mutations of the gene encoding CD40 ligand (CD40L; CD154). We report the clinical manifestations and mutational analysis of the CD40L gene observed in a male patient from a XHIM family. Having hypogammaglobulinemia and elevated IgM, the 3-yr-old boy exhibited the characteristic clinical features of XHIM. The patient suffered from frequent respiratory infections, and chronic enteritis caused by Cryptosporidium parvum. In addition, a lymph node biopsy and a culture from this sample revealed C. neoformans infection. Activated lymphocytes from the patient failed to express CD40L on their surface as assessed by flow cytometry and a missence mutation (W140R) was found at the XHIM hotspot in his CD40L cDNA to confirm the diagnosis. Genetic analysis of the mother and sister showed a heterozygote pattern, indicating carrier status. To our knowledge, this is the first report on the molecular diagnosis of an XHIM patient in Korea.

Expression of CD40 ligand, interferon-gamma and Fas ligand genes in endomyocardial biopsies of human cardiac allografts: correlation with acute rejection

The purpose of the present study was to investigate the expression (mRNA) of CD40 ligand (CD40L), interferon-gamma (IFN-gamma) and Fas ligand (FasL) genes in human cardiac allografts in relation to the occurrence of acute cardiac allograft rejection as well as its possible value in predicting acute rejection. The mRNA levels were determined by a semiquantitative reverse transcriptase-polymerase chain reaction method in 39 samples of endomyocardial biopsies obtained from 10 adult cardiac transplant recipients within the first six months after transplantation. Biopsies with ongoing acute rejection showed significantly higher CD40L, IFN-gamma and FasL mRNA expression than biopsies without rejection. The median values of mRNA expression in biopsies with and without rejection were 0.116 and zero for CD40L (P<0.003), 0.080 and zero for IFN-gamma (P<0.0009), and 0.156 and zero for FasL (P<0.002), respectively. In addition, the levels of IFN-gamma mRNA were significantly increased 7 to 15 days before the appearance of histological evidence of rejection (median of 0.086 in pre-rejection biopsies), i.e., they presented a predictive value. This study provides further evidence of heightened expression of immune activation genes during rejection and shows that some of these markers may present predictive value for the occurrence of acute rejection.