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  • 1
    Electronic Resource
    Electronic Resource
    p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload (2007)
    [s.l.] : Nature Publishing Group
    Nature 446 (2007), S. 444-448 
    Details
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function. However, prolonged cardiac hypertrophy causes heart failure, and its mechanisms are largely unknown. Here we show that cardiac angiogenesis is crucially involved in the adaptive mechanism of ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/nature05602
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  • 2
    Electronic Resource
    Electronic Resource
    A speedy method to detect inserted DNA fragment in cell lines transfected with retroviral vectors (2000)
    Springer
    Cytotechnology 34 (2000), S. 243-252 
    Details
    ISSN: 1573-0778
    Keywords: PCR ; quantitative PCR ; retroviral vector ; retroviral vectorspecific primer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Cells transfected by retroviral vectors are brought in agene of particular interest and are very useful in avariety of experiments. It is essential to testify that theDNA fragment was successfully introduced into the cellstogether with the retroviral vectors. Polymerase chainreaction is believed to be a fast and convenient method forthis purpose when using primers flanking the cloning siteof the inserted DNA. Unfortunately, a single PCR reactionoften fails to amplify the targeted fragment because of theexistence of endogenous virus DNA in cell genome. However,in this study we conducted a procedure for a single PCR,using vector-specific primers as well as a nested PCR, andsuccessfully detected the DNA fragments cloned in MFGretroviral vectors in 22 transfected cell lines. We alsoproved that real time quantitative PCR in combination withMFG-specific primer is useful to determine copy number ofthe retroviral vector in murine producer cell lines.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008187310534
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  • 3
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    An artificial virus-like nano carrier system: enhanced endosomal escape of nanoparticles via synergistic action of pH-sensitive fusogenic peptide derivatives (2008)
    Springer
    Details
    Publication Date: 2008-03-20
    Print ISSN: 1618-2642
    Electronic ISSN: 1618-2650
    Topics: Chemistry and Pharmacology
    Published by Springer
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  • 4
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    Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion (2005)
    American Society of Hematology
    Details
    Publication Date: 2005-07-15
    Description: Hepatic transdifferentiation of bone marrow cells has been previously demonstrated by intravenous administration of donor cells, which may recirculate to the liver after undergoing proliferation and differentiation in the recipient's bone marrow. In the present study, to elucidate which cellular components of human bone marrow more potently differentiate into hepatocytes, we fractionated human bone marrow cells into mesenchymal stem cells (MSCs), CD34+ cells, and non-MSCs/CD34- cells and examined them by directly xenografting to allylalcohol (AA)-treated rat liver. Hepatocyte-like cells, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), albumin (Alb), cytokeratin 19 (CK19), cytokeratin 18 (CK18), and asialoglycoprotein receptor (AGPR), and by reverse transcription-polymerase chain reaction (RT-PCR) for expression of AFP and Alb mRNA, were observed only in recipient livers with MSC fractions. Cell fusion was not likely involved since both human and rat chromosomes were independently identified by fluorescence in situ hybridization (FISH). The differentiation appeared to follow the process of hepatic ontogeny, reprogramming of gene expression in the genome of MSCs, as evidenced by expression of the AFP gene at an early stage and the albumin gene at a later stage. In conclusion, we have demonstrated that MSCs are the most potent component in hepatic differentiation, as revealed by directly xenografting into rat livers. (Blood. 2005;106:756-763)
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
    Published by American Society of Hematology
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  • 5
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    Soluble Factors Secreted from CD40 Ligand-Transfected Dendritic Cells Enhance TRAIL-Induced Apoptosis of Multiple Myeloma. (2004)
    American Society of Hematology
    Details
    Publication Date: 2004-11-16
    Description: Dendritic cells (DCs) belong to a family of antigen presenting cells that play a crucial role in the regulation of cellular and humoral immune responses to bacteria, virus and cancer. Recent studies also revealed that IFN-stimulated monocyte-derived DCs expressed functional TNF-related apoptosis-inducing ligand (TRAIL), suggesting that DCs may become cytotoxic effector cells to tumors. In this study, we investigate whether gene transfer of CD40-ligand (CD40L), that is known to be a potent stimulator in DC maturation, could induce cytotoxic activity of DCs or modulate susceptibility to TRAIL that is expressed on cytotoxic T and NK cells. Human monocyte-derived DCs infected with adenovirus vector encoding human CD40L (CD40L-DC) were induced to express superior higher levels of T-cell costimulatory molecules, DC maturation markers, MHC molecules and the production of IL-12 than soluble CD40L or TNF-alpha treated DCs. In addition to the phenotypic alternation of CD40L-DC, we found that cultivation of multiple myeloma cells ARH-77 with CD40L-DC significantly inhibited the expression of phospho-AKT and induced apoptosis in vitro. However, we could not detect the expression of TRAIL and Fas-ligand on CD40L-DC by flow cytometry, suggesting that these molecules are not involved. To examine whether soluble factors produced by CD40L-DC contribute to tumor cell apotosis, we cultured myeloma cells MM1S or ARH-77 with CD40L-DC in transwell plates. We found that myeloma cells in transwell culture were not lysed for 48 hours, but the susceptibility of apoptosis by recombinant TRAIL protein could be increased. To investigate soluble factors that could affect TRAIL sensitivity of myeloma cells, we next tested the effect of neutralizing antibodies specific for CD40L and TRAIL or recombinant IFN-gamma that might modulate TRAIL sensitivity. However, antibodies and recombinant protein did not affect, indicating that these factors might be not involved. Our findings suggest that CD40L-transduced DCs have the ability of induction of TRAIL susceptibility of multiple myeloma cells mediated by soluble factors that are currently investigated.
    Print ISSN: 0006-4971
    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
    Published by American Society of Hematology
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  • 6
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    A Novel Non-Cleavable Cell Surface Mutant of CD40-Ligand Induces Anti-Leukemic Immune Response and Prevent Systemic Inflammatory Reaction. (2004)
    American Society of Hematology
    Details
    Publication Date: 2004-11-16
    Description: CD40-ligand (CD40L), a member of the TNF family, is expressed transiently on activated CD4-positive T cells and mediates cognate interaction between T cell and antigen-presenting cell (APC) such as dendritic cells. We and other investigators have reported previously that transduction of human leukemia cells with adenovirus encoding full-length CD40-ligand resulted in upregulation of immune costimulatory molecules, enhance APC activity and generation of CTL to leukemia B cells. However, CD40L is cleaved to a soluble form (sCD40L) by metalloproteases and high levels of sCD40L may contribute to the systemic inflammatory diseases including systemic lupus erythematosus and rheumatoid arthritis, suggesting a potentially deleterious side effect of CD40L gene therapy. In this study we generated a non-cleavable mutant of CD40L to develop a potentially less toxic molecule for CD40L gene therapy. Four mutants of human CD40L (termed CD40Lm1, m2, m3 and m4) with point mutation of amino acids from E112 to P120 (suggested cleavage site) were created by RT-PCR and cloned into retrovirus and adenovirus vectors. These four mutants of CD40L were transduced into tumor cells and assessed sCD40L production by ELISA, demonstrating that all four mutants resulted in a fully non-cleavable mutant of CD40L. We also confirmed that CD40L mutants could stimulate CD40-positive B and dendritic cells and induce phenotypic alterations and IL-12 production. In order to examine systemic side effect of CD40L, we transplanted tumor cells expressing wild-type (CD40Lwt) or non-cleavable mutant of CD40L (CD40Lm3) in nude mice and have observed for one month period. Two weeks after transplantation, mice with tumors expressing CD40Lwt exhibited arthritis, systemic edema and slight diarrhea, but CD40Lm3 did not induce any systemic inflammatory effect. We also found increased plasma levels of sCD40L (〉800 pg/ml) in mice transplanted with CD40Lwt transfectant but not in CD40Lm3 transplanted mice. Additionally, mice with CD40Lwt resulted in increased number of infiltrating mononuclear cells in the liver and kidney, whereas no inflammatory cells were observed in the liver of mice with CD40Lm3. Overall, non-cleavable mutant of CD40L is fully capable of inducing immune response with less toxic molecule and useful tool for CD40L gene therapy of leukemia and lymphoma.
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    Electronic ISSN: 1528-0020
    Topics: Biology , Medicine
    Published by American Society of Hematology
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  • 7
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    Indian Hedgehog Stimulates Proliferation of Short-Term NOD/SCID-ß2mnull Repopulating Cells on Coculture with Human Stromal Cells. (2004)
    American Society of Hematology
    Details
    Publication Date: 2004-11-16
    Description: Hematopoietic stem cells (HSCs) represent a subset of bone marrow cells that are capable of self-renewal and of giving rise to all types of blood cells. However, the mechanisms involved in controlling the differentiation and self-renewal of HSCs remain largely unknown. The Indian hedgehog (Ihh) signal has been shown to play an essential role in inducing hematopoietic tissue during embryogenesis. We investigated the roles of the Ihh in postnatal hematopoiesis using coculture system between CD34+ cells and human stromal cells. Ihh gene transfer into hTERT-stromal cells enhanced the expression of BMP4, angiopoietin-1 and Wnt5A, and their hematopoietic supporting potential was elevated compared with control stromal cells, as indicated by the colony-forming units in culture (CFU-C, 26±2 vs. 59±3-fold of the initial cell number; CFU-Mix, 63±37 vs. 349±116). Engraftments of NOD/SCID-ß2mnull repopulating cells (RCs) at 8 weeks post-transplantation expanded on Ihh-stromal cells were significantly higher compared with control coculture results and engraftments were neutralized by addition of an anti-hedgehog antibody. Limiting dilution analysis indicated that SCID RCs proliferated more efficiently on Ihh-stromal cells than those on control stromal cells. The degree of expansion on Ihh-stromal cells was estimated to be 6.6-fold greater than that of control stromal cells, according to the Poisson predicted frequency. However, engraftment of NOD/SCID-ß2mnull RCs was decreased by 13 weeks post-transplantation of hematopoietic cells that had been expanded on either Ihh-stromal cells or control stromal cells, although engraftment of human hematopoietic cells that were expanded on Ihh-stromal cells is higher than that for cells expanded on control stromal cells (%CD45+ cells: 3.28±0.60 vs. not detected). These results suggest that Ihh-stromal cells are quite potent to support human short-term RCs in NOD/SCID-ß2mnull mice. Thus, hedgehog signaling is considered to play an important role in the hematopoietic microenvironment.
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    Topics: Biology , Medicine
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  • 8
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    Selective and Efficient Gene Delivery of CD40-Ligand by a Fiber-Modified Adenovirus Vector with Specific Antibody to Human Leukemia and Myeloma. (2004)
    American Society of Hematology
    Details
    Publication Date: 2004-11-16
    Description: The use of adenovirus vector for cancer gene therapy is limited by their low transduction efficiency for lymphoma, leukemia and myeloma. We previously reported that highly efficient gene delivery of CD40-ligand by a modified adenovirus vector with the integrin-binding motif, RGD, in the H1 loop of the fiber knob (AxCAhCD40L-F/RGD) could induce phenotypic alteration followed by T cell immune response to autologous leukemia cells. But the utility of adenovirus with RGD-motif is still limited by their lack of specificity on tumor cells. Recent studies revealed a novel strategy of targeting adenovirus using a bispecific single-chain antibody (scFv) specific for adenovirus and target molecules on tumor cell surface. However, this approach should permit the production of high quantities of active bispecific scFv for in vivo use. To target adenovirus to hematopoietic tumor cells efficiently, we herein constructed a modified adenovirus vector that contained a synthetic immunoglobulin G-binding domain (termed Z33) in H1 loop of the fiber knob. A recombinant adenovirus encoding EGFP, lacZ (as reporter gene; Ax3CAZ3-F/Z33 or Ax3EGFP-F/Z33) or CD40L (as a therapeutic gene; Ax3CD40L-F/Z33) with Z33-modified fiber were tested for gene transfer efficiency into human tumors such as lymphoma, leukemia and myeloma cells. By the treatment with various antibodies specific for CD20 (Rituximab), CD40, CD38, CCR2 or CXCR4 that are expressed on leukemic cells, we achieved 3 to 10-fold enhancement of gene expression in lymphoma/leukemia (Ramos, Daudi or THP-1) and myeloma cells (MM1S, IM-9 or KMS5) compared with control IgG-treated tumors. We also examined specific gene delivery to freshly isolated leukemia B cells from patients that also contains normal lymphocytes. By using antibody to CD20 or CD40, we could selectively deliver CD40L gene with Ax3CD40L-F/Z33 into leukemia B cells (〉50% at 300 pu/cell), but not in T and monocytes, followed by the induction of immune costimulatory molecules that are important in anti-leukemia immune response. Overall, our results indicated that combination of Z33-modified adenovirus vector and tumor specific antibody can be used as a modality for the gene therapy of leukemia and myeloma.
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  • 9
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    Adenoviral Vector-Mediated Transfer of the Indian Hedgehog Gene Modulates Lymphomyelopoiesis In Vivo. (2007)
    American Society of Hematology
    Details
    Publication Date: 2007-11-16
    Description: Indian hedgehog (Ihh) plays an essential role in angiogenesis, hematogenesis and epiphysis formation during embryogenesis. In the present study, we injected an adenoviral vector (Adv) carrying the mock-control (adv-control) or Ihh gene (Adv-Ihh) into SCID or BALB/c mice to evaluate the effects of lhh on the regulation of postnatal hematopoiesis in vivo. After the intravenous injection of Adv-Ihh, the expression of vector-derived Ihh mRNA was detected in the liver. Four weeks after administration of Adv-Ihh to SCID mice, we observed an increase in the number of c-Kit+ cells and clonogenic cells/105 mononuclear cells in the bone marrow as compared with adv-control administrated mice. Moreover, after administration of Adv-Ihh to BALB/c mice, the number of splenic B220+IgMlowCD23intCD21int B lymphocytes (Figure 1) and CD4+ T lymphocytes (Figure 2) was strongly increased. Furthermore, the number of thymic double negative (DN)2, DN3, CD8+ immature single positive and CD4+/CD8− cells was significantly elevated relative to the number in mice that received the control Adv vector. Our results suggest that enhanced signaling by Ihh can modulate the proliferation and differentiation of splenic B lymphocytes and thymic T lymphocytes during BM hematopoiesis in vivo. Thus, modulation of the Hh signaling pathway may provide a therapeutic strategy to stimulate lymphomyelopoiesis in vivo. Figure Figure Figure Figure
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  • 10
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    Wnt3/RhoA/ROCK Signaling Pathway Is Involved in VLA6-Dependent Adhesion-Mediated Drug Resistance of Multiple Myeloma. (2007)
    American Society of Hematology
    Details
    Publication Date: 2007-11-16
    Description: Adhesion of myeloma cells to BM stromal cells is now considered to play a critical role in chemo-resistance. However, little is known about the molecular mechanism of cell adhesion mediated drug resistance (CAM-DR) in multiple myeloma. In this study, we focused on relationship between drug resistance and expression of Wnts, the factor regulating the cell adhesion and proliferation, in myeloma cells. To gain insight into involvement of Wnt signaling in CAM-DR, we first screened the expression of Wnt family in myeloma cell lines (RPMI8226, ARH77, KMS-5 and MM1S) by reverse transcription-polymerase chain reaction analysis. Although the mRNAs of Wnt2b, Wnt7a and Wnt10b were variably expressed in some of myeloma cell lines, Wnt3 mRNA was detected in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human BM stromal cells and accumulation of β-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPMI8226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human BM stromal cells. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against Doxorubicin. This CAM-DR was significantly reduced by anti-integrinβ1 antibody, anti- integrinα6 antibody and a Wnt-receptor competitor, secreted Frizzled related protein-1 and Rho kinase inhibitor (Y27632 and OH-fasudil), but not by the specific inhibitor of canonical signaling (DKK-1), indicating that Wnt-mediated CAM-DR which is dependent on integrinα6/β1 (VLA-6)-mediated attachment to stromal cells is induced by Wnt/RhoA-Rho kinase (ROCK) pathway signal. This CAM-DR for doxorubicin was also significantly reduced by Wnt3 siRNA transfer to KMS-5 and further augmented by addition of Wnt3 conditioned medium. These results indicate that Wnt3 contributes to VLA-6-mediated CAM-DR via the Wnt/RhoA/ROCK pathway of myeloma cells. Thus, the Wnt3/RhoA/ROCK signaling pathway could be a promising molecular target to overcome CAM-DR.
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