Caspase-9 (Casp-9) induces death signals by triggering other types of caspase activation, and its expression greatly influences the onset of apoptosis. During the isolation of apoptosis-related genes involved in glucocorticoid (GC)-induced cell death in murine thymic lymphomas, we found that the antisense gene of the transcription factor activator protein-4 (AP-4) inhibited dexamethasone-induced apoptosis. Western blot analysis revealed that the expression of Bcl-x L , Bax, and Apaf-1 was not affected in cells transfected with sense or antisense AP-4 genes. In contrast, both the expression and activation of Casp-9 were inhibited in the antisense AP-4 transfectants. We isolated the 2.4-kb 5-flanking region of Casp-9, and the promoter activity was investigated. We found the AP-4-binding sites at ؊1.55 and ؊1.38 kb to be responsible for the promoter activity. Furthermore, a negative cis-element was expected to exist between bases ؊1140 and ؊944. When the cells were treated with dexamethasone, a rapid down-regulation of AP-4 and Casp-9 was observed whether the cells were GC-sensitive lymphomas or GC-insensitive L929 fibroblast cells. In addition, L929 cells pretreated with dexamethasone were found to be resistant to subsequent treatment with etoposide, an apoptosis-inducing reagent. GC has a two-sided effect on apoptosis, i.e. a pro-apoptotic effect on certain cell types and a prosurvival effect on other cell types. Our findings will explain, at least in part, this effect.
A CD4+CD8+ double-positive thymocyte cell line, 257-20-109 was established from BALB/c mice thymocytes and used to analyze the requirements to induce CD4 or CD8 single-positive (SP) T cells. CD4SP cells were induced from 257-20-109 cells by anti-CD3 stimulation in the presence of the FcR-positive macrophage cell line, P388D1. During stimulation, maturation events, such as the down-regulation of CD24 and the up-regulation of CD69, H-2Dd, CD5, and Bcl-2, were recognized. Furthermore, these CD4SP cells appeared to be functional because the cells produced IL-2 and IL-4 when activated with phorbol ester and calcium ionophore. In contrast, CD8SP cells could be induced by stimulation with fixed anti-CD3 after removal of stimulation. To investigate the extent of signals required for CD4SP and CD8SP, the cells stimulated under either condition for 2 days were sorted and transferred to different culture conditions. These results suggested that the fate of lineage commitment was determined within 2 days, and that CD4 lineage commitment required longer activation. Furthermore, the experiments with subclones of 257-20-109 demonstrated that the lower density of CD3 did not shift the cells from CD4SP to CD8SP, but only reduced the amount of CD4SP cells. In contrast, when the 257-20-109 cells were stimulated by the combination of fixed anti-CD3 and anti-CD28, the majority of the cells shifted to CD4SP, with an enhancement of extracellular signal-regulated kinase 1 phosphorylation. Our results indicate that the signals via TCR/CD3 alone shifted the double-positive cells to CD8SP cells, but the reinforced signals via TCR/CD3 and costimulator could commit the cells to CD4SP.
Background Limited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage. Methods Full-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, −AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-β were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared. Results Superficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-β was associated with higher migratory activity of the superficial-zone chondrocytes. Conclusions In enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-β combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.
We studied some aspects of the quantitative and qualitative features of heterologous recombinant (re) virus-vector-induced, antigen-specific CD8 + T cells against Trypanosoma cruzi. We used three different, highly-attenuated re-viruses, i.e., influenza virus, adenovirus and vaccinia virus, which all expressed a single, T. cruzi antigen-derived CD8 + T cell epitope. The use of two out of three vectors or the triple virus vector vaccination regimen not only confirmed that the re-vaccinia virus, which was placed last in order for sequential immunization, was an effective booster for the CD8 + T cell immunity in terms of the number of antigen-specific CD8 + T cells, but also demonstrated that i) the majority of cells exhibit the effector memory (T EM ) phenotype, ii) robustly secrete IFN-γ, iii) express higher intensity of the CD122 molecule and iv) present protective activity against T. cruzi infection. In contrast, placing the re-influenza virus last in sequential immunization had a detrimental effect on the quantitative and qualitative features of CD8 + T cells. The triple virus vector vaccination was more effective at inducing a stronger CD8 + T cell immunity than using two re-viruses. The different quantitative and qualitative features of CD8 + T cells induced by different immunization regimens Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access
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