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BACKGROUND: Although current immunotherapeutic strategies including adenovirus (AdV)-mediated gene therapy and dendritic cell (DC) vaccine can all stimulate antitumor cytotoxic T lymphocyte (CLT) responses, their therapeutic efficiency has still been limited to generation of prophylactic antitumor immunity against re-challenge with the parental tumor cells or growth inhibition of small tumors in vivo. However, it is the well-established tumors in animal models that mimic clinical patients with existing tumor burdens. Alpha tumor necrosis factor (TNF-alpha) is a multifunctional and immunoregulatory cytokine that induces antitumor activity and activates immune cells such as DCs and T cells. We hypothesized that a combined immunotherapy including gene therapy and DC vaccine would have some advantages over each modality administered as a monotherapy. METHODS: We investigated the antitumor immunotherapeutic efficiency of gene therapy by intratumoral injection of AdVTNF-alpha and DC vaccine using subcutaneous injection of TNF-alpha-gene-engineered DC(TNF-alpha) cells, and further developed a combined AdV-mediated TNF-alpha-gene therapy and TNF-alpha-gene-engineered DC(TNF-alpha) vaccine in combating well-established MO4 tumors expressing the ovalbumin (OVA) gene in an animal model. RESULTS: Our data show that vaccination of DC(TNF-alpha) cells pulsed with the OVA I peptide can (i) stimulate type 1 immune response with enhanced antitumor CTL activities, (ii) induce protective immunity against challenge of 5 x 10(5) MO4 tumor cells, and (iii) reduce growth of the small (3-4 mm in diameter), but not large, established MO4 tumors (6-8 mm in diameter). Our data also show that AdVTNF-alpha-mediated gene therapy can completely eradicate small tumors in 6 out of 8 (75%) mice due to the extensive tumor necrosis formation, but not the large tumors (0%). Interestingly, a combined AdVTNF-alpha-mediated gene therapy and TNF-alpha-gene-engineered DC(TNF-alpha) vaccine is able to cure 3 out of 8 (38%) mice bearing large MO4 tumors, indicating that the combined immunotherapy strategy is much more efficient in combating well-established tumors than monotherapy of either gene therapy or DC vaccine alone. CONCLUSIONS: This novel combined immunotherapy may become a tool of considerable conceptual interest in the implementation of future clinical objectives. 相似文献
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Regulation of soluble and surface-bound TRAIL in human T cells, B cells, and monocytes 总被引：8，自引：0，他引：8
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF/nerve growth factor superfamily that, apart from inducing cell death in susceptible cells, displays immunoregulatory functions influencing, for instance, T cell proliferation. It can be found in two forms: membrane-bound and soluble protein. The regulation of these is still not fully understood. In this study, we have analyzed the regulation of TRAIL surface expression and secretion in human T cells, B cells, and monocytes in response to specific stimuli. T cells, B cells, and monocytes were cultured in the presence of phytohemagglutinin (PHA)+interleukin (IL-2), anti-CD40+IL-4, and lipopolysaccharide (LPS), respectively. In particular, not only PHA+IL-2 but also LPS were able to induce secretion of soluble TRAIL, but did not enhance the expression of surface-bound TRAIL. Simultaneously, we investigated the effect of the pleiotropic stimulus interferon (IFN)-beta, known to target all leukocyte subsets, on TRAIL. Predominantly, monocytes were affected by IFN-beta, causing both release of soluble TRAIL and upregulation of the surface-bound form. IFN-beta, however, did not cause any upregulation of TRAIL in T cells. Our data serve as a basis to better understand the complex regulation of TRAIL in human peripheral immune cells and might help to clarify the role of the TRAIL system in immunopathology. 相似文献
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Ishigaki H Yamamoto Y Ishida H Kajino K Itoh Y Fujiyama Y Ogasawara K 《Microbiology and immunology》2006,50(8):655-662
Tumor-infiltrating stroma cells (TISC) as well as tumors themselves are thought to be involved in tumor-related immunosuppression, which is one of the critical mechanisms of tumor escape from immune surveillance. However, preparation of TISC is difficult because of the small proportion of TISC in established tumors. Thus, the cells thought to be involved in tumor-related immunosuppression are generally prepared from spleens or draining lymph nodes in tumor-bearing mice. In this study, we developed a method for directly preparing TISC from established tumors in order to analyze their function. Using green fluorescent protein (GFP) transgenic (Tg) mice and C57BL/6 mice transplanted with bone marrow (BM) cells of GFPTg mice, we detected three subpopulations of TISC: one is compatible with immature myeloid cells (ImC) derived from BM and the two other subpopulations, CD11b(+) cells and CD11b(-) cells, do not originate from BM. The TISC including these subpopulations but not each subpopulation independently after culturing with tumors in the presence of GM-CSF could suppress T cell proliferation induced by anti-CD3. In our system, tumors did not inhibit T cell responses directly, but unknown factors from tumors affected immunosuppression by TISC. 相似文献
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Yoshino N Ami Y Someya K Ando S Shinohara K Tashiro F Lu Y Honda M 《Microbiology and immunology》2000,44(5):363-372
A simian/human immunodeficiency virus (SHIV)-NM3n containing the human nef, but not the monkey nef, and vpr genes of SIV was inoculated into two cynomolgus monkeys, resulting in systemic infection with a minimum level of transient virus load. In order to study the nature of immune responses associated with the prevention of a pathogenic SHIV, the SHIV-NM3n-inoculated monkeys and three naive monkeys were intravenously challenged with a pathogenic SHIV containing the envelope gene of HIV-1 89.6. After the heterologous virus challenge, all of the SHIV-NM3n-inoculated animals completely avoided the loss of CD4+ T lymphocytes in PBMC as well as lymphoid tissues compared to pathogenic SHIV-injected control animals. The inhibition of CD4+ cell depletion was associated with maintaining the proliferative response of helper T-cells against SIV p27 in the previously nonpathogenic virus-inoculated animals following the pathogenic virus challenge. Furthermore, the decline of CD28+ cells, the increase in CD95+ cells, and the enhancement of in vitro apoptosis in PBMC were inhibited in the non-pathogenic virus-inoculated animals. These results suggest that nonpathogenic SHIV-NM3n infection induces the protection of monkeys from heterologous pathogenic viruses that may be associated with blocking the change in immune responses and the cell loss induced by a pathogenic virus. 相似文献
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Sommer G Weise S Kralisch S Scherer PE Lössner U Blüher M Stumvoll M Fasshauer M 《Journal of cellular biochemistry》2008,104(6):2241-2247
Serum amyloid A (SAA) 3 has been characterized as an inflammatory adipocyte-secreted acute-phase reactant. In the current study, regulation of SAA3 by the proinflammatory and insulin resistance-inducing cytokine interleukin (IL)-1beta was determined in 3T3-L1 and brown adipocytes. Interestingly, SAA3 mRNA and protein synthesis were dramatically increased by IL-1beta in a time-dependent fashion with maximal induction after 24 h. Furthermore, IL-1beta significantly induced SAA3 mRNA expression dose-dependently with maximal 36.4-fold upregulation seen at 2 ng/ml effector. Moreover, IL-1beta-induced SAA3 expression was mediated by nuclear factor-kappaB and janus kinase 2. Taken together, our data show a potent upregulation of SAA3 by IL-1beta. 相似文献
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Michelle A. Dunstone Lars Kjer‐Nielsen Lyudmila Kostenko Anthony W. Purcell Andrew G. Brooks Jamie Rossjohn James McCluskey 《Acta Crystallographica. Section D, Structural Biology》2004,60(8):1425-1428
Human CD3 is an essential multisubunit complex that plays a fundamental role in T‐cell signalling, T‐cell development and surface expression of the αβ T‐cell receptor. The CD3 complex comprises the CD3ɛγ and CD3ɛδ heterodimers and the CD3ζζ homodimer. Here, the expression of the human CD3ɛγ and CD3ɛδ heterodimers, both of which were expressed as single‐chain polypeptides, is reported. Following refolding, functional heterodimers were immunoaffinity purified from improperly folded heterodimers using OKT3, a therapeutic monoclonal antibody specific for the CD3ɛ chain. Subsequently, the Fab fragment of OKT3 was used to complex individually with the CD3ɛγ and CD3ɛδ heterodimers. Crystals of scCD3ɛγ–FabOKT3 were grown using 15%(w/v) PEG 3350, 200 mM potassium fluoride, 100 mM Tris–HCl pH 8.0. Crystals of scCD3ɛδ–FabOKT3 were grown using 20%(w/v) PEG 3350, 200 mM potassium formate, 100 mM Tris–HCl pH 8.0, 2%(v/v) MPD. Crystals of both complexes diffract to beyond 3 Å resolution. scCD3ɛγ–FabOKT3 crystals belonged to space group P21, with unit‐cell parameters a = 67.70, b = 55.77, c = 96.05 Å, β = 100.85° and one complex per asymmetric unit. scCD3ɛδ–FabOKT3 crystals belong to space group P21, with unit‐cell parameters a = 101.67, b = 50.36, c = 138.7 Å, β = 108.84°, suggesting two complexes per asymmetric unit. 相似文献
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Over the past 30 years, the beneficial therapeutic effects of selected low energy, time varying electromagnetic fields (EMF) have been documented with increasing frequency to treat therapeutically resistant problems of the musculoskeletal system. However, the underlying mechanisms at a cellular level are still not completely understood. In this study, the effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMF) on osteoclastogenesis, cultured from murine bone marrow cells and stimulated by 1,25(OH)(2)D(3), were examined. Primary bone marrow cells were cultured from mature Wistar rats and exposed to ELF-PEMF stimulation daily for 7 days with different intensities of induced electric field (4.8, 8.7, and 12.2 micro V/cm rms) and stimulation times (0.5, 2, and 8 h/day). Recruitment and authentication of osteoclast-like cells were evaluated, respectively, by determining multinuclear, tartrate resistant acid phosphatase (TRAP) positive cells on day 8 of culture and by the pit formation assay. During the experiments, cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and prostaglandin-E(2) (PGE(2)) were assayed using the enzyme linked immunosorbent assay (ELISA). These findings suggest that ELF-PEMF can both enhance (approximately 50%) and suppress (approximately 27%) the formation of osteoclast-like cells in bone marrow culture, depending on the induced electric field intensity. In addition, consistent correlations were observed between TNF-alpha, IL-1beta, and osteoclast-like cell number after exposure to different induced electric field intensities of ELF-PEMF. This in vitro study could be considered as groundwork for in vivo ELF-PEMF clinical applications on some osteoclast-associated bone diseases. 相似文献
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Hanan A. Al-Nazal Emily Cooper Mei Fong Ho Sharareh Eskandari Victoria Majam Ashwini Kumar Giddam Waleed M. Hussein Md. Tanjir Islam Mariusz Skwarczynski Istvan Toth Sanjai Kumar Ali Zaid Michael Batzloff Danielle I. Stanisic Michael F. Good 《Cell host & microbe》2021,29(6):894-903.e5
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Langnaese K Richter K Smalla KH Krauss M Thomas U Wolf G Laube G 《Developmental neurobiology》2007,67(4):422-437
Knock out mice deficient for the splice-isoform alphaalpha of neuronal nitric oxide synthase (nNOSalphaalpha) display residual nitric oxide synthase activity and immunosignal. To attribute this signal to the two minor neuronal nitric oxide synthase splice variants, betabeta and gammagamma, we generated isoform-specific anti-peptide antibodies against the nNOSalphaalpha specific betabeta-finger motif involved in PDZ domain scaffolding and the nNOSbetabeta specific N-terminus. The nNOSalphaalpha betabeta-finger-specific antibody clearly recognized the 160-kDa band of recombinant nNOSalphaalpha on Western blots. Using immunocytochemistry, this antibody displayed, in rats and wild-type mice, a labeling pattern similar to but not identical with that obtained using a commercial pan-nNOS antibody. This similarity indicates that the majority of immunocytochemically detectable nNOS is not likely to be complexed with PDZ-domain proteins via the betabeta-finger motif. This conclusion was confirmed by the inhibition of PSD-95/nNOS interaction by the nNOSalphaalpha betabeta-finger antibody in pull-down assays. By contrast, nNOSalphaalpha betabeta-finger labeling was clearly reduced in hippocampal and cortical neuropil areas enriched in NMDA receptor complex containing spine synapses. In nNOSalphaalpha knock out mice, nNOSalphaalpha was not detectable, whereas the pan-nNOS antibody showed a distinct labeling of cell bodies throughout the brain, most likely reflecting betabeta/gammagamma-isoforms in these cells. The nNOSbetabeta antibody clearly detected bacterial expressed nNOSbetabeta fusion protein and nNOSbetabeta in overexpressing HEK cells by Western blotting. Immunocytochemically, individual cell bodies in striatum, cerebral cortex, and in some brain stem nuclei were labeled in knock out but not in wild-type mice, indicating an upregulation of nNOSbetabeta in nNOSalphaalpha deficient animals. 相似文献
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Non-proteolytic group 2 allergen, Der p 2 (DP2) is known as a major allergen derived from house dust mite Dermatophagoides pteronyssinus.Paracellular epithelial barrier, being composed of a number of tight junction (TJ) molecules, plays pivotal roles in resistance of pathogen invading. However, whether DP2 affects epithelial TJ molecules is unclear. Therefore, we aimed to investigate the effects of DP2 on epithelial TJ molecules, and the mechanism by which expression of junction molecules is regulated by DP2. Cell cycle and mRNA expression of TJ proteins of lung alveolar cell A549 were analyzed by RT-PCR and flow cytometry. Level of claudin-2, subcellular distribution of b-catenin and kinase activation was determined using immunoblot. Our findings revealed that DP2 had no significant influence on cell cycle distribution but affected mRNA expression of TJ molecules including claudin-2, occludin, and ZO-1 in A549 cells. Our results showed that DP2 significantly elevated level of claudin-2 and increased expression and nuclear translocation of b-catenin. Moreover, DP2 enhanced the phosphorylation of glycogen synthase kinase-3b (GSK-3b) and its potential upstream regulator Akt. The DP2-induced claudin-2 expression was also suppressed by GSK-3b inhibitor (lithium chloride) and phosphatidyl inositol 3-phosphate kinase (PI3K) inhibitor (wortamannin). Taken together, these findings showed that DP2 increased claudin-2 expression and its cell surface distribution in A549 cells, which may attribute to phosphorylation of GSK-3b and Akt and the consequent increase and nuclear translocation of b-catenin. It is suggested that presence of DP2 may alter epithelial junction by regulating expression of TJ molecules. 相似文献
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Molecular profiling of CD8 T cells in autochthonous melanoma identifies Maf as driver of exhaustion 
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下载免费PDF全文 Marilyn Giordano Coralie Henin Julien Maurizio Claire Imbratta Pierre Bourdely Michel Buferne Lukas Baitsch Laurent Vanhille Michael H Sieweke Daniel E Speiser Nathalie Auphan‐Anezin Anne‐Marie Schmitt‐Verhulst Grégory Verdeil 《The EMBO journal》2015,34(15):2042-2058
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Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) resulting in the inhibition of GSK-3 kinase activity and increase of beta-catenin levels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulation of these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell-cell and cell-matrix adhesion. Moreover, increase in cell-cell adhesion but not cell-matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells. 相似文献
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This study was performed to elucidate the possible mechanism of the disturbance of hemopoiesis by HCMV infection. Saos-2 cells constitutively express mRNA of GM-CSF, and its expression was profoundly decreased by HCMV infection, which required full replication of the virus and was mediated by soluble factors released from the HCMV-infected Saos-2 cells. TGF-beta1 production was statistically and significantly increased from one day after HCMV infection. Expression and production of GM-CSF in Saos-2 cells were restored when a culture supernatant of HCMV-infected Saos-2 cells was reacted with neutralizing anti-TGF-beta antibody. Conclusively, HCMV inhibits GM-CSF expression in Saos-2 cells partly by the increased production of TGF-beta1. 相似文献
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Hesling C Fattet L Teyre G Jury D Gonzalo P Lopez J Vanbelle C Morel AP Gillet G Mikaelian I Rimokh R 《EMBO reports》2011,12(7):665-672
TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT. 相似文献
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Zhou S 《Journal of cellular biochemistry》2011,112(6):1651-1660
Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs. 相似文献