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Unstimulated Cells (unstimulated + cell)
Selected AbstractsTransgene-activated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cellsTHE JOURNAL OF GENE MEDICINE, Issue 1 2006Jung Park Abstract Background Adult primary mesenchymal cells of different origin which can be obtained with minor donor site morbidity are considered for articular cartilage repair. This study aims at a comparison of their chondrogenic potential. Methods Mesenchymal cells were isolated from perichondrium/periosteum, bone marrow or fat of adult rats and found to be positive for the stem-cell-related antigens Sca-1, c-Kit, CD10, CD13 and CD90 by reverse transcription polymerase chain reaction (RT-PCR). Chondrogenic differentiation was induced by applying recombinant bone morphogenetic protein-2 (BMP-2) or adenoviral vectors carrying BMP-2 cDNA, followed by micromass culture. The stimulated cells were characterized by RT-PCR, cell proliferation and apoptosis assays. Expression of aggrecan, collagen type I, II, IX and X and alkaline phosphatase genes was analyzed by RT-PCR, immunofluorescence and immunohistochemistry in comparison with unstimulated control cells. Adenovirally stimulated cells were transplanted into mechanically generated partial-thickness cartilage lesions in the patellar groove of the rat femur. Quality and integration of the repair tissues were assessed by histochemical and immunohistochemical methods. Results Stimulation with BMP-2 or AdBMP-2 led to an up-regulation of cartilage-specific gene expression in all three cell populations studied, most rapidly and prominently in the perichondrial/periosteal cells, which showed a 3200-fold increase of type II collagen mRNA and reached the highest absolute levels of type II and IX collagen transcripts after stimulation. Similar results were obtained for the bone marrow stromal cells (BMSC), while the respective transcript levels in fat stromal cells declined after an initial more than 30-fold elevation. Following transplantation in vivo, AdBMP-2-infected perichondrial/periosteal cells produced a proteoglycan-rich, type II collagen-positive matrix with only faint staining for type I collagen. The repair tissue originating from AdBMP-2-infected BMSC showed less intense type II collagen staining, but a relatively proteoglycan-rich matrix, weakly positive for type I collagen. Transgene-activated fat stromal cells formed rather fibrous tissue mainly composed of type I collagen. Unstimulated cells of the three different populations gave only rise to fibrous tissue. Conclusions Perichondrium/periosteum-derived cells and BMSC seem superior to cells isolated from fat with respect to forming hyaline cartilaginous tissue. A chondrogenic stimulus, e.g. by transfer of BMP-2 cDNA, appears to be required for initiation and support of chondrogenic differentiation. Copyright © 2005 John Wiley & Sons, Ltd. [source] Differential expression of mast cell characteristics in human myeloid cell linesEXPERIMENTAL DERMATOLOGY, Issue 9 2004Pia Welker Abstract:, In order to better understand the mechanisms governing display of mast cell characteristics in human myeloid cells, we have studied the mast cell phenotype in human promyelocytic (HL-60) and myelocytic (U-937, TPH-1) vs. basophilic (KU-812) and mast cell (HMC-1) lines, in part also in skin mast cells and blood monocytes, at mRNA and protein level before and after stimulation with mast cell growth factors. In unstimulated cells, mRNA for the stem cell factor (SCF) receptor c-kit and the gamma chain of the high-affinity IgE receptor (Fc,RI) was noted in all cells studied. Like mast and basophilic cells, THP-1 cells expressed the Fc,RI, and , chains and weakly histidine decarboxylase (HDC), but they lacked mRNA for mast cell-specific proteases [tryptase, chymase, carboxypeptidase A (CPA)]. In contrast, HL-60 and U-937 cells lacked Fc,RI,, but expressed tryptase and chymase, HL-60 cells also CPA. KU-812 cells failed to express the basophil-specific marker 2D7. After a 10-day culture with SCF or fibroblast supernatants, baseline mRNA expression of most mast cell characteristics was upregulated, whereas c-kit mRNA expression decreased in all but THP-1 cells. Differential mRNA expression of Fc,RI vs. protease (tryptase) was confirmed at protein level by immunocytochemistry and enzymatic activity. KU-812 cells are thus closest to skin mast cells in that they express all molecules studied, except for chymase, followed by THP-1 cells that lack all mast cell proteases. In contrast, HL-60 and U-937 cells fail to express the Fc,RI, and , chains but express most mast cell proteases. The selective and differential expression of mast cell characteristics in human myeloid cell lines suggests that induction of the mast cell phenotype is regulated by several independent genes and that mast cells and basophils branch off at early and distinct points of myeloid development. [source] Lead-dependent effects on arachidonic acid accumulation and the proliferation of vascular smooth muscleJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2002Robert V. Dorman Abstract Lead (Pb2+) has been implicated in the development of hypertension and atherosclerosis. The proliferation of vascular smooth muscle cells (VSMC) is a central feature of both conditions and there is evidence that Pb2+ potentiates serum-dependent cell growth. The aim of this work was to examine the role of phospholipase A2 in mitogen-dependent VSMC proliferation and determine if Pb2+ interacts with this system in order to potentiate mitotic events. It was observed that cell proliferation induced by angiotensin II, or fetal bovine serum, required the activation of a Ca2+ -dependent cytosolic phospholipase A2 and the subsequent release of unesterified arachidonic acid. This path was affected by Pb2+ as the metal increased the amount of arachidonic acid accumulation induced by either mitogen. In addition, Pb2+ potentiated mitogen-induced DNA synthesis when present at lower doses (0.02 or 0.2 mg%), but had no effect on DNA synthesis, or cell numbers, in unstimulated cells. However, a high dose (2 mg%) of Pb2+ attenuated the DNA synthesis stimulated by angiotensin II, or serum, but induced the accumulation of unesterified arachidonic acid in unstimulated cells. A biphasic effect of Pb2+ on cell numbers and viability was also observed as 0.02 or 0.2 mg% Pb2+ did not affect cell numbers or trypan blue exclusion in unstimulated cells, while 2 mg% Pb2+ reduced cell numbers and viability. It appeared, therefore, that the lower concentrations of Pb2+ increased arachidonic acid release and DNA synthesis only in stimulated VSMC, perhaps due to further activation of a Ca2+ -dependent processes. In contrast, the high dose of Pb2+ reduced DNA synthesis in stimulated cells and reduced cell numbers and viability in unstimulated cells, which may relate to the noted increase in unesterified arachidonic acid. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:245,253, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10045 [source] Different cellular localization, translocation, and insulin-induced phosphorylation of PKB, in HepG2 cells and hepatocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2002Noor Afshan Syed Abstract Protein kinase B (PKB), a serine/threonine protein kinase, prevents apoptosis and promotes cellular transformation. PKB activity is stimulated by insulin. In this report, we examined the relative amounts of expression, location, and translocation upon insulin stimulation of PKB, in normal primary hepatocytes and carcinoma cells, HepG2 cells. Non-phosphorylated PKB, was present in both types of unstimulated cells. The phosphorylated form of the enzyme was present in the nucleus of unstimulated HepG2 cells but not in normal hepatocytes. In the cytoplasm, PKB, was found in greater abundance in the hepatocytes as compared in HepG2 cells. Insulin induced the translocation of phosphorylated PKB, from the nucleus to the nuclear membrane in HepG2 cells. In contrast, insulin caused translocation and phosphorylation of PKB, from the cytosol to the plasma membrane in normal hepatocytes. In addition, there is a higher expression of PKB, in the HepG2 cells as compared to normal primary hepatocytes. These findings provide an important distinction between hepatocellular HepG2 cells and normal liver cells and suggest that the presence of constitutively active nuclear PKB in the transformed cells might be an important contributor in cell transformation and immortality of hepatoma cells. J. Cell. Biochem. 86: 118,127, 2002. © 2002 Wiley-Liss, Inc. [source] Effects of statins on microgliaJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2005Catharina Lindberg Abstract High serum cholesterol level has been shown as one of the risk factors for Alzheimer's disease (AD), and epidemiological studies indicate that treatment with cholesterol-lowering substances, statins, may provide protection against AD. An acute-phase reaction and inflammation, with increased levels of proinflammatory cytokines, are well known in the AD brain. Notably, there is evidence for antiinflammatory activities of statins, such as reduction in proinflammatory cytokines. Consequently, it is of interest to analyze the effects of statins on microglia, the main source of inflammatory factors in the brain, such as in AD. The aims of this study were to determine the effects of statins (atorvastatin and simvastatin) on microglial cells with regard to the secretion of the inflammatory cytokine interleukin-6 (IL-6) and cell viability after activation of the cells with bacterial lipopolysaccharides (LPS) or ,-amyloid1,40 (A,1,40) and in unstimulated cells. Cells of the human microglial cell line CHME-3 and primary cultures of rat neonatal cortical microglia were used. Incubation with LPS or A,1,40 induced secretion of IL-6, and A,1,40, but not LPS, reduced cell viability. Both atorvastatin and simvastatin reduced the basal secretion of IL-6 and the cell viability of the microglia, but only atorvastatin reduced LPS- and A,1,40 -induced IL-6 secretion. Both statins potentiated the A,1,40 -induced reduction in cell viability. The data indicate the importance of also considering the microglial responses to statins in evaluation of their effects in AD and other neurodegenerative disorders with an inflammatory component. © 2005 Wiley-Liss, Inc. [source] The large form of ADAR 1 is responsible for enhanced hepatitis delta virus RNA editing in interferon- , -stimulated host cellsJOURNAL OF VIRAL HEPATITIS, Issue 3 2006D. Hartwig Summary., Hepatitis delta virus (HDV) RNA editing controls the formation of hepatitis-delta-antigen-S and -L and therefore indirectly regulates HDV replication. Editing is thought to be catalysed by the adenosine deaminase acting on RNA1 (ADAR1) of which two different forms exist, interferon (IFN)- , -inducible ADAR1-L and constitutively expressed ADAR1-S. ADAR1-L is hypothesized to be a part of the innate cellular immune system, responsible for deaminating adenosines in viral dsRNAs. We examined the influence of both forms on HDV RNA editing in IFN- , -stimulated and unstimulated hepatoma cells. For gene silencing, an antisense oligodeoxyribonucleotide against a common sequence of both forms of ADAR1 and another one specific for ADAR1-L alone were used. IFN- , treatment of host cells led to approximately twofold increase of RNA editing compared with unstimulated controls. If ADAR1-L expression was inhibited, this substantial increase in editing could no longer be observed. In unstimulated cells, ADAR1-L suppression had only minor effects on editing. Inhibition of both forms of ADAR1 simultaneously led to a substantial decrease of edited RNA independently of IFN- , -stimulation. In conclusion, the two forms of ADAR1 are responsible almost alone for HDV editing. In unstimulated cells, ADAR1-S is the main editing activity. The increase of edited RNA under IFN- , -stimulation is because of induction of ADAR1-L, showing for the first time that this IFN-inducible protein is involved in the base modification of replicating HDV RNA. Thus, induction of ADAR1-L may at least partially cause the antiviral effect of IFN- , in natural immune response to HDV as well as in case of therapeutic administration of IFN. [source] CXC and CC chemokines induced in human renal epithelial cells by inflammatory cytokinesAPMIS, Issue 7 2009ELISKA THORBURN (NEE KRASNA) Human renal epithelial cells might play an important role during the allograft rejection by producing chemokines in response to proinflammatory cytokines such as tumor necrosis factor (TNF)-, and interleukin (IL)-1, produced by endothelial and epithelial cells early after transplantation. The production of chemokines allows inflammatory cells to be drawn into the kidney graft and therefore plays a critical role in the pathophysiologic processes that lead to the rejection of renal transplant. In this process, two chemokine superfamilies, the CC and the CXC chemokines, are the most important. The CC chemokines target mainly monocytes and T lymphocytes, while most of the CXC chemokines attract neutrophils. We showed in our study that in vitro, in unstimulated cells, basal mRNA expression of CXC chemokines (Gro,, Gro,, Gro,, ENA-78 and GCP-2, IL-8) that attract neutrophils was detectable and expression of these genes and chemokine release were increased in TNF-,- and IL-1,-induced renal epithelial cells. Most of the CC chemokines [monocyte chemotactic protein-1 (MCP-1), macrophage Inflammatory protein 1 beta (MIP-1,), regulated upon activation, normal T cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP-3,)] showed detectable mRNA expression only after stimulation with proinflammatory cytokines and not in control cells. TNF-, seems to induce preferably the expression of RANTES, MCP-1, interferon-inducible protein (IP-10) and Interferon-Inducible T-cell Alpha Chemoattractant (I-TAC), while IL-1, induces mainly IL-8 and epithelial neutrophil-activating peptide 78 (ENA-78). [source] Up-regulated cytokine-inducible SH2-containing protein expression in allergen-stimulated T cells from hen's egg-allergic patientsCLINICAL & EXPERIMENTAL ALLERGY, Issue 9 2008Y. Nakajima Summary Background Although changes in the fine balance of allergen-specific T cells are crucial in the pathogenesis of allergic diseases, their roles in the allergic reaction to hen's eggs (HE) have not yet been fully analysed. Objective Using microarray technology, allergen-stimulated T cells from HE-allergic children were analysed to identify genes that are specifically up-regulated in these cells. Methods RNA from CD4+ CD14, cells, fractionated from allergen-stimulated peripheral mononuclear cells, was analysed using a whole -genome microarray and real-time RT-PCR. The protein expression of selected genes was ascertained by flow cytometry. Results In microarray analyses of allergen-stimulated T cells, 43 genes were up-regulated in HE-allergic children but not in non-HE-allergic children. Among these, up-regulation of three genes, cytokine -inducible SH2-containing protein (CISH), nuclear factor of , light polypeptide gene enhancer in B-cell inhibitor Z (NFKBIZ) and B-cell CLL/lymphoma 2 (BCL2), was confirmed by real-time quantitative RT-PCR. CISH, but not NFKBIZ or BCL2, showed a significantly higher ratio of antigen-stimulated cell transcription over unstimulated cells in HE-allergic than in non-HE-allergic children (P<0.01). Flow-cytometric analysis revealed that the percentage of CD25+CISH+ cells in CD4+ cells from patients with HE allergy was significantly higher than that in controls (P<0.01). The expression level of CISH was significantly higher in IL-4+ Th2 cells than in IFN-,+ Th1 cells. Conclusion We noted that CISH expression in allergen-stimulated CD4+ T cells from HE-allergic patients was significantly increased in both mRNA and protein levels compared with that from non-HE-allergic children. [source] | |||||||||||||