Nuclear Factor-κB (NF-κB) is a major transcription regulator of immune response, apoptosis and cell-growth control genes, and is upregulated in inflammatory bowel disease (IBD), both ulcerative ...colitis (UC) and Crohn's disease. The NFKB1 gene encodes the NF-κB p105/p50 isoforms. Genome-wide screens in IBD families show evidence for linkage on chromosome 4q where NFKB1 maps. We sequenced the NFKB1 promoter, exon 1 and all coding exons in 10 IBD probands and two controls, and identified six nucleotide variants, including a common insertion/deletion promoter polymorphism (−94ins/delATTG). Using pedigree-based transmission disequilibrium tests, we observed modest evidence for linkage disequilibrium (LD), independent of linkage, between the −94delATTG allele and UC in 131 out of 235 IBD pedigrees with UC offspring (P=0.047–0.052). This allele was also more frequent in the 156 non-Jewish UC probands from the 235 IBD pedigrees than in 149 non-Jewish controls (P=0.015). The −94delATTG association with UC was replicated in a second set of 258 unrelated, non-Jewish UC cases and 653 new, non-Jewish controls (P=0.021). Nuclear proteins from normal human colon tissue and colonic cell lines, but not ileal tissue, showed significant binding to −94insATTG but not to −94delATTG containing oligonucleotides. NFKB1 promoter/exon 1 luciferase reporter plasmid constructs containing the −94delATTG allele and transfected into either HeLa or HT-29 cell lines showed less promoter activity than comparable constructs containing the −94insATTG allele. Therefore, we have identified the first potentially functional polymorphism of NFKB1 and demonstrated its genetic association with a common human disease, ulcerative colitis.
Because blood cells can be obtained with relatively easy and safe procedure, they have been routinely used for transfusion and transplantation purposes. And they are now considered as attractive cell ...sources for developing new gene therapies including cancer therapy using various immune cells, and regenerative therapy using hematopoietic stem cells or induced pluripotent stem cells (iPS) cells. For example, chimeric antigen receptor modified autologous T cells have been considered as effective therapy for various cancers. And iPS cells have been easily established from peripheral T cells for the purpose of treating various diseases. However, in spite of these possibilities, the development of the safer and more efficient genetic modification methods of hematopoietic cells is imminent. In this study, we developed the novel measles viral (MV) vector which enables us to transduce multiple genes into immune cells. The wild type measles virus is one of the aerosol-transmitted viruses and has strong infectious capacity to immune cells, and epithelial cells via signaling lymphocyte activation molecule (SLAM) or nectin-4. First, we modified the wild type measles virus genome to non-transmissible and non-lytic, and equipped with the ability of transducing multiple genes, at most six genes, into target cells. Briefly, the intrinsically non-segmented wild type virus genome was divided into two segments and point mutations were introduced into the virus genes encoding hemagglutinin and the matrix protein. Moreover, as the fusion protein gene was removed from the virus genome, the virus could not replicate in neighbor cells. We examined the gene transduction efficiency of the gene modified measles virus (H8-Fd-MV vector) into hematopoietic cells. We first constructed the H8-Fd-MV vector with GFP gene and transduced into hematopoietic progenitor cells and immune cells from human cord blood and peripheral blood. We observed that almost all of HPCs from cord blood (99.7% in floating cells expressed CD34), T cells (99.9% in CD3+ cells), and B cells (98.2% in CD19+ cells) from peripheral blood expressed GFP at two days after the transduction. Especially, to express GFP gene in human peripheral T cells, it was not necessary to pre-stimulate them with CD3/CD28 beads (99.6% in stimulating T cells (72.9% in SLAM+ cells) v.s. 82.6 % in non-stimulating cells (37.4% in SLAM+ cells)). T cells from cord blood showed almost all naïve phenotype (CD4+ cells: 93.2±1.8% in CD45RA+CCR7+ cells, 1.7±1.1% in CD45RA+CCR7- cells; CD8+ cells: 41.6±5.7% in CD45RA+CCR7+ cells, and 41.9±11.0% in CD8+CD45RA+CCR7- cells) and T cells transduced by MV vector expressed GFP more (CD4+ cells: 80.3±13.7%, and CD8+ cells: 82.5±8.5%) than those transduced by Sendai viral vector (CD4+ cells: 15.5±0.7%, and CD8+ cells: 17.4±5.4%). These data suggested that H8-Fd-MV vector could transduce GFP gene efficiently into various T cell lineages including naïve T cells, which had been difficult to be transduced with classical gene transduction methods. We next generated H8-Fd-MV vector for expressing 6 genes (OCT4, SOX2, KLF4, L-MYC, PIN1, and GFP) and transduced into stimulated T cells. After 3 days, GFP+ T cells expressed all of these 6 genes. We also detected that more than 50% of the stimulated T cells with IL-2 expressed GFP at 14 days after the transduction. After 27 days from transfection, embryonic stem cell (ES cell)-like colonies were picked up and analyzed the character. These cells showed ES cell morphology over 20 times passages and expressed pluripotent marker (NANOG, OCT4, Tra-1-60, Tra-1-81). We also found T cell receptor rearrangements in these cells. Embryoid bodies from these cells expressed three germ line markers in vitro. We next examined the hematopoietic differentiation of these cells using coculture system with murine embryonic aorta-gonad-mesonephros region-derived stromal cell line (AGM-3 cells). The co-cultured cells harvested at day 12 expressed CD34 and CD45. These data suggested that we established iPS cells from terminal differentiated T cells using H8-Fd-MV vector for expressing reprogramming factor.
These results indicated that multiple genes were expressed efficiently in immune cells using H8-Fd-MV vector. Highly efficient transduction ability of MV vector for T cells would enable us to develop new gene therapy targeting cancer using gene modified T cells as well as organ regeneration using iPS cells.
No relevant conflicts of interest to declare.
Interstitial cells of Cajal (ICC) are believed to lnitlate the basic contractile activity of the gastrointestlnal tract. Interstitial cells of Cajal express c‐kit receptor tyroslne kinase and are ...deficient in Ws/Ws mutant rats with a small deletion of the c‐kit gene. As Ws/Ws rats show remarkable bile reflux to the stomach, the contraction pressure of the pylorus was compared between Ws/Ws and control +/+ rats. The contraction pressure of the pylorus was measured using a mlcrotransducer, which was Inserted through a pln‐hole in the anterlor wall of the stomach under anesthesla. The magnitude of bile reflux was estimated by measurlng the content of bile acids In the stomach. The c‐kit messenger RNA‐expressing cells were detected by in sltu hybrldlzatlon. Frequency and the maxlmum pressure of the contractlon were comparable between Ws/Ws and +/+ rats, but the duration of the contractlon was significantly shorter In Ws/Ws rats than In +/+ rats. The number of c‐kit messenger RNA‐expresslng ICC in the pylorus of Ws/Ws rats was 1.7% that of +/+ rats. The bile reflux observed in Ws/Ws rats was attributed to the decrease in the duration of the pyloric contraction, which appeared to result from the deficlency of c‐kit messenger RNA‐expressing ICC.
The Fas/CD95/APO-1 ligand (FasL) is a death cytokine that binds to cell surface Fas/CD95/APO-1 receptor, yet a possible role of FasL expression in p53-dependent apoptosis is not fully understood in ...many human malignancies, including renal carcinoma.
By Northern blot and Western blot analyses, we determined the effect of p53 on the FasL and Fas receptor expression. To do this, we employed an in vitro renal carcinoma model system that was previously established by stably co-transfecting a temperature-sensitive mutant allele of the p53 tumor suppressor (ts-p53) with either the c-Myc oncogene or adenovirus E1A oncogene in baby rat kidney (BRK) epithelial cells. The ts-p53 is activated only at a permissive temperature. The transactivation activity of p53 was assessed by luciferase reporter assays. The sub-G1 cell population in the cell cycle representing apoptotic cell death was measured by flow cytometric analysis.
We found that the level of endogenous FasL, but not Fas receptor, was increased at a permissive temperature with delayed kinetics when compared with p21WAF1 expression, but was coincident with p53-induced apoptosis, whereas an apoptosis-defective mutant p53, which lacks the PxxP region (P: Proline, x: any amino acid), failed to induce FasL expression and hence apoptosis. Notably, p53-induced apoptosis was completely blocked by overexpressing a dominant negative inhibitor of the FADD/Mort-1, a pro-apoptotic adaptor that lies immediately downstream of the FasL/Fas receptor.
These results suggest that the FasL is a critical downstream effector of p53-dependent apoptosis in a cultured BRK renal carcinoma model system.
1 Department of Kinesiology, 2 Department of Cell Biology and Molecular Genetics, and 3 Department of Public and Community Health, University of Maryland, College Park; 4 Department of Biochemistry ...and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland; 5 Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, and Division of Cardiology, Emory University, Atlanta, Georgia; 6 The Harvey M. and Lyn P. Meyerhoff Inflammatory Bowel Disease Center, Gastroenterology Division, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; and 7 Department of Kinesiology, College of Health Professions, Temple University, Philadelphia, Pennsylvania
Submitted 12 February 2007
; accepted in final form 18 July 2007
In endothelial cells, NF- B is an important intracellular signaling molecule by which changes in wall shear stress are transduced into the nucleus to initiate downstream endothelial nitric oxide synthase ( NOS3 ) gene expression. We investigated whether NF- light-chain gene enhancer in B cells 1 (NFKB1) promoter polymorphism ( –94 NFKB1 I/D, where I is the insertion allele and D is the deletion allele) was associated with 1 ) NOS3 gene expression in endothelial cells under physiological levels of unidirectional laminar shear stress (LSS) and 2 ) endothelial function in prehypertensive and stage I hypertensive individuals before and after a 6-mo supervised endurance exercise intervention. Competitive EMSAs revealed that proteins present in the nuclei of endothelial cells preferentially bound to the I allele NFKB1 promoter compared with the D allele. Reporter gene assays showed that the I allele promoter had significantly higher activity than the D allele. In agreement with these observations, homozygous II genotype cells had higher p50 expression levels than homozygous DD genotype cells. Cells with the homozygous II genotype showed a greater increase in NOS3 protein expression than did homozygous DD genotype cells under LSS. Functional experiments on volunteers confirmed higher baseline reactive hyperemic forearm blood flow, and, furthermore, the subgroup analysis revealed that DD homozygotes were significantly less prevalent in the exercise responder group compared with II and ID genotypes. We conclude that the –94 NFKB1 I/D promoter variation contributes to the modulation of vascular function and adaptability to exercise-induced flow shear stress, most likely due to differences in NFKB1 gene transactivity.
nuclear factor- light-chain gene enhancer in B cells 1; nitric oxide synthase 3; shear stress
Address for reprint requests and other correspondence: M. D. Brown, Dept. of Kinesiology, College of Health Professions, Temple Univ., 1800 N. Broad St., Pearson Hall, Rm. 129, Philadelphia, PA 19122 (e-mail: brownmd{at}temple.edu )
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