Biomedical subjects

Antonio Bernad

Publications and source records attributed to Antonio Bernad.

At least 19 recordsLinked to original sources

PubMed2006

SOCS up-regulation mobilizes autologous stem cells through CXCR4 blockade.

The chemokine CXCL12 influences self-renewal and differentiation of hematopoietic stem cell precursors in bone marrow by directing them toward specific stromalcell components. CXCL12 up-regulates members of the SOCS family through JAK/STAT activation, a mechanism that attenuates chemokine responses. SOCS expression may thus modulate retention of hematopoietic precursors (Sca-1(+) c-Kit(+)Lin(-) cells) in bone marrow. We show that in bovine growth hormone transgenic mice and in growth hormone-treated mice, SOCS up-regulation correlated with a large number of Sca-1(+) c-Kit(+)Lin(-) cells in blood. Retroviral transduction of SOCSs blocked in vitro migration of Sca-1(+)c-Kit(+)Lin(-) cells, as well as their capacity to reconstitute lethally irradiated mice. Furthermore, in lethally irradiated mice reconstituted with bone marrow infected by a tetracycline-regulated, SOCS-expressing lentiviral vector, doxycycline treatment promoted rapid, extensive precursor mobilization to the periphery. The results indicate that by blocking CXCR4-mediated functions, SOCSs modulate hematopoietic precursor cell retention in bone marrow, and suggest the therapeutic interest of SOCS manipulation in several pathologic situations.

Animals↗

PubMed2006

Inducible model for beta-six-mediated site-specific recombination in mammalian cells.

The prokaryotic beta recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in chromatin-integrated substrates. Here, we demonstrate that an enhanced green fluorescent protein (EGFP)-fused version of beta recombinase (beta-EGFP) is fully active, retaining most specific activity. It is used to develop a recombination-dependent activatable gene expression (RAGE) system based on the androgen receptor (AR) ligand-binding domain (LBD). Two hybrid molecules, a direct fusion of the LBD-AR to the C-terminus of beta recombinase (beta-AR) and a triple fusion of beta-EGFP to the same ligand-binding domain (beta-EGFP-AR), were engineered and their subcellular behavior, stability and catalytic activity were evaluated. Both chimeric beta recombinase proteins showed in vivo inducible recombinogenic activity dependent on addition of an androgen receptor agonist, although the beta-AR fusion protein demonstrated more accurate ligand-dependent translocation from cytoplasm to nucleus.

Androgens↗

PubMed2006

Generation of GABAergic and dopaminergic interneurons from endogenous embryonic olfactory bulb precursor cells.

During the embryonic period, many olfactory bulb (OB) interneurons arise in the lateral ganglionic eminence (LGE) from precursor cells expressing Dlx2, Gsh2 and Er81 transcription factors. Whether GABAergic and dopaminergic interneurons are also generated within the embryonic OB has not been studied thoroughly. In contrast to abundant Dlx2 and Gsh2 expression in ganglionic eminences (GE), Dlx2 and Gsh2 proteins are not expressed in the E12.5-13.5 mouse OB, whereas the telencephalic pallial domain marker Pax6 is abundant. We found GABAergic and dopaminergic neurons originating from dividing precursor cells in E13.5 OB and in short-term dissociated cultures prepared from the rostral half of E13.5 OB. In OB cultures, 22% of neurons were GAD+, of which 53% were Dlx2+, whereas none expressed Gsh2. By contrast, 70% of GAD+ cells in GE cultures were Dlx2+ and 16% expressed Gsh2. In E13.5 OB slices transplanted with EGFP-labeled E13.5 OB precursor cells, 31.7% of EGFP+ cells differentiated to GABAergic neurons. OB and LGE precursors transplanted into early postnatal OB migrated and differentiated in distinct patterns. Transplanted OB precursors gave rise to interneurons with dendritic spines in close proximity to synaptophysin-positive boutons. Interneurons were also abundant in differentiating OB neural stem cell cultures; the neurons responded to the neurotrophin Bdnf and expressed presynaptic proteins. In vivo, the Bdnf receptor TrkB colocalized with synaptic proteins at the glomeruli. These findings suggest that, in addition to receiving interneurons from the LGE, the embryonic OB contains molecularly distinct local precursor cells that generate mature GABAergic and dopaminergic neurons.

Animals↗

PubMed2005

Nucleocytoplasmic shuttling of STK16 (PKL12), a Golgi-resident serine/threonine kinase involved in VEGF expression regulation.

PKL12/STK16 protein is the first identified mammalian member of a ser/thr kinase subfamily that is conserved across several kingdoms, with a broad expression pattern in murine tissues and cell types. Endogenous STK16 subcellular localization was evaluated by indirect immunofluorescence in NIH/3T3 and NRK cells, demonstrating a Golgi-associated pattern that appears to be independent of signals provided by integrin pathways. When cells were treated with brefeldin A (BFA) or nocodazole, drugs that promote Golgi disorganization, we observed STK16 translocation to the nuclear compartment. Constitutive overexpression of this protein by retroviral vectors also promotes accumulation of STK16 in the nuclear compartment, as shown by subfractionation studies. A kinase-dead STK16 mutant (E202A) was used to demonstrate that both the Golgi association and the nuclear translocation capabilities seem to be independent of the STK16 kinase activity. In addition, we show that STK16 overexpression in several cell lines enhances their capacity to produce and secrete VEGF. To confirm these data in vivo, we injected tumor cells overexpressing STK16 into immunodeficient BALBc/SCID mice. HT1080-derived tumors overexpressing STK16 showed increased volume and number of blood vessels compared to controls. Altogether, these data concur with previous reports suggesting a potential role for STK16 as a transcriptional co-activator.

Active Transport, Cell Nucleus↗

PubMed2005

ZAP-70 upregulation in transformed B cells after early pre-BI cell transplant into NOD/SCID mice.

Understanding of the signal transduction pathways that lead to B cell development is of extreme interest to learn how alterations in these pathways might initiate malignant transformation. Long-term cultured early pre-BI cells can differentiate into IgM+ B cells after transplant into NOD/SCID mice, offering the possibility to explore checkpoints in B cell development. Using DNA microarray and Western blot analysis of IgM+ B cells vs parental early pre-BI cells, we demonstrated that zeta-associated protein 70 (ZAP-70) is upregulated in our B cell differentiation model. Unlike parental ZAP-70- early pre-BI cells, ZAP-70+ IgM+ B cells exhibited a transformed phenotype, as indicated by BCL-6 expression, a high Ki-67 proliferation index, resistance to IL-7 deprivation-induced apoptosis, and an increased repopulation rate in NOD/SCID mice. These data show the characterization and generation of a novel murine leukemia model with many similarities to human ZAP-70+ B cell chronic lymphocytic leukemia.

Animals↗

PubMed2005

Spontaneous human adult stem cell transformation.

Human adult stem cells are being evaluated widely for various therapeutic approaches. Several recent clinical trials have reported their safety, showing them to be highly resistant to transformation. The clear similarities between stem cell and cancer stem cell genetic programs are nonetheless the basis of a recent proposal that some cancer stem cells could derive from human adult stem cells. Here we show that although they can be managed safely during the standard ex vivo expansion period (6-8 weeks), human mesenchymal stem cells can undergo spontaneous transformation following long-term in vitro culture (4-5 months). This is the first report of spontaneous transformation of human adult stem cells, supporting the hypothesis of cancer stem cell origin. Our findings indicate the importance of biosafety studies of mesenchymal stem cell biology to efficiently exploit their full clinical therapeutic potential.

Adipose Tissue↗

PubMed2005

Polymerase mu is up-regulated during the T cell-dependent immune response and its deficiency alters developmental dynamics of spleen centroblasts.

Mammalian DNA polymerase mu (Polmu), preferentially expressed in secondary lymphoid organs, is shown here to be up-regulated in germinal centers after immunization. Alternative splicing appears to be part of Polmu regulation during an immune response. We generated Polmu-deficient mice that are viable and show no anatomical malformation or serious alteration in lymphoid populations, with the exception of an underrepresentation of the B cell compartment. Young and aged homozygous Polmu(-/-) mice generated similar immune responses after immunization with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gammaglobulin (CGG), compared with their wild-type littermates. Nonetheless, the kinetics of development of the centroblast population showed significant differences. Hypermutation analysis of the rearranged heavy chain intron region in centroblasts isolated from NP-CGG-immunized Polmu(-/-) mice showed a similar quantitative and qualitative somatic mutation spectrum, but a lower representation of heavily mutated clones. These results suggest that although it is not a critical partner, Polmu modulates the in vivo somatic hypermutation process.

Animals↗

PubMed2005

Wiskott-Aldrich syndrome protein is needed for vaccinia virus pathogenesis.

Smallpox, caused by variola virus, was a devastating disease in humans, but how the virus evolved a strategy to spread to tissue remains unknown. Through the use of microarrays, we identified the gene encoding the Wiskott-Aldrich syndrome protein (WASP), one of the five known WASP family members, which has been induced in the course of infection of human cells with different strains of vaccinia virus (VV) (S. Guerra, L. A. Lopez-Fernandez, A. Pascual-Montano, M. Munoz, K. Harshman, and M. Esteban, J. Virol. 77:6493-6506, 2003; S. Guerra, L. A. Lopez-Fernandez, R. Conde, A. Pascual-Montano, K. Harshman, and M. Esteban, J. Virol. 78:5820-5834, 2004). In a mouse model, we evaluated the role of WASP in infection with VV, a close relative of variola virus. WASP(-/-) (KO) mice infected intranasally and intraperitoneally with VV showed reduced weight loss and mortality compared to wild-type (WT) mice. WASP expression correlated with VV replication in the ovaries but not in the liver or spleen. WT mouse macrophages express WASP but not N-WASP; after VV infection, WASP levels increase threefold. KO macrophages lack N-WASP expression and, when VV infected, are incapable of inducing actin tails and producing extracellular virus. These functions were rescued in KO macrophages after ectopic WASP expression. Overall, our findings demonstrate that WASP has a role in orthopoxvirus infections. Use of WASP proteins for virus spread via the actin tail provides a selective advantage for VV, and probably variola virus, dissemination to distant tissues.

Actins↗

PubMed2004

Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate.

DNA polymerase mu (Pol mu) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol mu protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol mu in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol mu to mutation of G and C residues during SHM. In vitro analysis of Pol mu misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol mu to induce transient template/primer misalignments.

B-Lymphocytes↗

PubMed2004

The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer.

Several non-hypercalcemic analogs of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) show antitumor activity in a subset of cancer patients. High vitamin D receptor (VDR) expression, which is associated with good prognosis but is lost during tumor progression. We show that the SNAIL transcription factor represses VDR gene expression in human colon cancer cells and blocks the antitumor action of EB1089, a 1,25(OH)(2)D(3) analog, in xenografted mice. In human colon cancers, elevated SNAIL expression correlates with downregulation of VDR.

Animals↗

PubMed2004

Dedifferentiated adult articular chondrocytes: a population of human multipotent primitive cells.

OBJECTIVE: To test the hypothesis that dedifferentiated adult human cartilage chondrocytes (HAC) are a true multipotent primitive population. METHODS: Studies to characterize dedifferentiated HAC included cell cycle and quiescence analysis, cell fusion, flow-FISH telomere length assays, and ABC transporter analysis. Dedifferentiated HAC were characterized by flow cytometry, in parallel with bone marrow mesenchymal stem cells (MSC) and processed lipoaspirate (PLA) cells. The in vitro differentiation potential of dedifferentiated HAC was studied by cell culture under several inducing conditions, in multiclonal and clonal cell populations. RESULTS: Long-term HAC cultures were chromosomically stable and maintained cell cycle dynamics while showing telomere shortening. The phenotype of dedifferentiated HAC was quite similar to that of human bone marrow MSC. In addition, this population expressed human embryonic stem cell markers. Multiclonal populations of dedifferentiated HAC differentiated to chondrogenic, osteogenic, adipogenic, myogenic, and neurogenic lineages. Following VEGF induction, dedifferentiated HAC expressed characteristics of endothelial cells, including AcLDL uptake. A total of 53 clonal populations of dedifferentiated HAC were efficiently expanded; 17 were able to differentiate to chondrogenic, osteogenic, and adipogenic lineages. No correlation was observed between telomere length or quiescent population and differentiation potential in the clones assayed. CONCLUSION: Dedifferentiated HAC should be considered a human multipotent primitive population.

Adipocytes↗

PubMed2004

Gold glyconanoparticles as new tools in antiadhesive therapy.

Gold glyconanoparticles (GNPs) have been prepared as new multivalent tools that mimic glycosphingolipids on the cell surface. GNPs are highly soluble under physiological conditions, stable against enzymatic degradation and nontoxic. Thereby GNPs open up a novel promising multivalent platform for biological applications. It has recently been demonstrated that specific tumor-associated carbohydrate antigens (glycosphingolipids and glycoproteins) are involved in the initial step of tumor spreading. A mouse melanoma model was selected to test glyconanoparticles as possible inhibitors of experimental lung metastasis. A carbohydrate-carbohydrate interaction is proposed as the first recognition step for this process. Glyconanoparticles presenting lactose (lacto-GNPs) have been used successfully to significantly reduce the progression of experimental metastasis. This result shows for the first time a clear biological effect of lacto-GNPs, demonstrating the potential application of this glyconanotechnology in biological processes.

Animals↗

PubMed2004

Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with a median survival below the age of 20 due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous hematopoietic stem cells or T cells could represent an alternative treatment applicable to all patients. We investigated whether WAS gene transfer with MMLV-based oncoretroviral and HIV-based lentiviral vectors could restore normal functions of patients' T cells. T cells transduced either with lentiviral vectors expressing the WAS protein (WASP) from the ubiquitous PGK promoter or the tissue-specific WASP promoter or with an oncoretroviral vector expressing WASP from the LTR, reached normal levels of WASP with correction of functional defects, including proliferation, IL-2 production, and lipid raft upregulation. Lentiviral vectors transduced T cells from WAS patients at higher rates, compared to oncoretroviral vectors, and efficiently transduced both activated and naive WAS T cells. Furthermore, a selective growth advantage of T cells corrected with the lentiviral vectors was demonstrated. The observation that lentiviral vector-mediated gene transfer results in correction of T cell defects in vitro supports their application for gene therapy in WAS patients.

Antibodies↗

PubMed2004

Absence of hematopoiesis from transplanted olfactory bulb neural stem cells.

Neural stem cells giving rise to neurons and glia cells have been isolated from the embryonic and adult central nervous system. The extent to which they are able to differentiate into cells of non-neural lineages, such as the hematopoietic lineage, is nonetheless unclear. We previously reported the isolation of stem cells from the mouse olfactory bulb neuroepithelium. In the present study, we analysed whether olfactory bulb stem cells (OBSC) can generate cells with hematopoietic features. Cells were prepared from the olfactory bulbs of transgenic mice expressing enhanced green fluorescent protein (EGFP). In culture, transgenic cells proliferated with the same kinetics as wild-type cells. Following mitogen removal, both cell types gave rise to similar numbers of neurons, astrocytes and oligodendrocytes, indicating that EGFP overexpression does not alter OBSC proliferation and differentiation patterns. When these cells were injected into the tail vein of irradiated mice, no hematopoietic cells derived from the OBSC could be recovered in their peripheral blood, spleen or bone marrow. By contrast, when OBSC were transplanted into the adult brain, EGFP-positive cells were found in the striatum and corpus callosum; differentiated cells expressed antigenic markers of neurons and astrocytes. These results suggest that embryonic olfactory bulb stem cells are not endowed with the potential to produce hematopoiesis.

Animals↗

PubMed2003

Interleukin-6 deficiency affects bone marrow stromal precursors, resulting in defective hematopoietic support.

Interleukin-6 (IL-6) is a critical factor in the regulation of stromal function and hematopoiesis. In vivo bromodeoxyuridine incorporation analysis indicates that the percentage of Lin(-)Sca-1(+) hematopoietic progenitors undergoing DNA synthesis is diminished in IL-6-deficient (IL-6(-/-)) bone marrow (BM) compared with wild-type BM. Reduced proliferation of IL-6(-/-) BM progenitors is also observed in IL-6(-/-) long-term BM cultures, which show defective hematopoietic support as measured by production of total cells, granulocyte macrophage-colony-forming units (CFU-GMs), and erythroid burst-forming units (BFU-Es). Seeding experiments of wild-type and IL-6(-/-) BM cells on irradiated wild-type or IL-6-deficient stroma indicate that the hematopoietic defect can be attributed to the stromal and not to the hematopoietic component. In IL-6(-/-) BM, stromal mesenchymal precursors, fibroblast CFUs (CFU-Fs), and stroma-initiating cells (SICs) are reduced to almost 50% of the wild-type BM value. Moreover, IL-6(-/-) stromata show increased CD34 and CD49e expression and reduced expression of the membrane antigens vascular cell adhesion molecule-1 (VCAM-1), Sca-1, CD49f, and Thy1. These data strongly suggest that IL-6 is an in vivo growth factor for mesenchymal precursors, which are in part implicated in the reduced longevity of the long-term repopulating stem cell compartment of IL-6(-/-) mice.

Animals↗

PubMed2003

A comparison of targeting performance of oncoretroviral versus lentiviral vectors on human keratinocytes.

The epidermis, like other rapidly renewing tissues, relies on a stem cell compartment to undergo constant regeneration. In order to develop realistic and long-lasting therapeutic approaches for some skin disorders, gene transfer to these critical cells must be obtained. While efficient retroviral ex vivo targeting and transgene integration in human keratinocytes is tightly dependent on proliferation, transferring genetic information to quiescent cells in culture also presents advantages, including the possibility of targeting putative dormant epidermal stem cells. In the present study we compared the efficiency of transduction achieved with a third-generation of human immunodeficiency virus (HIV)-based lentiviral vector to that obtained with a Moloney murine leukemia oncoretroviral vector (MLV) on proliferating and quiescent human keratinocytes growing in vitro in standard Rheinwald and Green cultures as well as in confluent organotypic cultures. Each viral vector contained the enhanced green fluorescent protein (EGFP) as a reporter gene. The lentiviral vector, but not the MLV vector, led to EGFP expression both in nondividing and proliferating epidermal cell populations in vitro. This feature was clearly evident when direct targeting of human keratinocytes, forming part of the epidermal component of an organotypic skin culture, was attempted. Keratinocytes modified by both MLV and the lentiviral vector allowed long-term regeneration of genetically engineered human skin on the backs of immunodeficient nonobese diabetic/severe combined immunodeficiency disorders (NOD/SCID) mice. However, EGFP transgene expression in the context of the MLV (long-terminal repeat [LTR]-driven) or lentiviral vector (cytomegalovirus [CMV]-driven) demonstrated clear differences both in quantitative terms and in the in vivo localization pattern.

Animals↗

PubMed2003

Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background.

Human neo-organ formation from stem cells can only be assayed by in vivo xenotransplantation. The human nonobese diabetic-severe combined immunodeficient (HuNOD/scid) CD34+ cell transplantation is a model that allows examination of hematopoietic tissue formation, although human hematopoietic cell maturation is abortive. Conventional humanization of the cytokine microenvironment has depended on generation of human cytokine-transgenic mice in strains appropriate for conventional plasmid microinjection, followed by backcrossing, a costly and time-consuming approach. Lentiviral vector infection of single-cell embryos was recently reported to produce transgenic animals. Using this approach, we have generated direct human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic mice from lentivirus-microinjected NOD/scid embryos, with 68% efficiency and 100% penetrance; this allowed us to obtain NOD/scid transgenic mice with considerable savings of resources. This powerful technique should assist in producing novel mouse models for the study of human blood cell lineage development and other human neo-organs from stem cell xenotransplantation for which a similar "humanization" rationale may be required.

Animals↗

PubMed2003

Selective inactivation of p27(Kip1) in hematopoietic progenitor cells increases neointimal macrophage proliferation and accelerates atherosclerosis.

Excessive proliferation of immune cells and vascular smooth myocytes (VSMCs) contributes to atherosclerosis. We have previously shown that whole-body inactivation of the growth suppressor p27 exacerbates atherosclerosis in apolipoprotein E-null mice (apoE-/-), and this correlated with increased proliferation of arterial macrophages and VSMCs. In the present study, we postulated that targeted disruption of bone marrow (BM) p27 is sufficient to enhance arterial macrophage proliferation and atherosclerosis. To test this hypothesis, sublethally irradiated apoE-/- mice with an intact p27 gene received a BM transplant from either apoE-/- or p27-/-apoE-/- doubly deficient donor mice and challenged with a high-cholesterol diet. Compared with mice that received an apoE-/- BM transplant, reconstitution with p27-/-apoE-/- doubly deficient marrow increased the expression of proliferating cell nuclear antigen in neointimal macrophages and accelerated aortic atherosclerosis, and this correlated with augmented aortic expression of the inflammatory cytokines CCL2/MCP-1 (monocyte chemoattractant protein 1) and CCL5/RANTES (regulated on activation, normal T-cell expressed and secreted). Overall, these findings provide evidence that p27 deficiency in hematopoietic progenitor cells enhances the inflammatory/proliferative response induced by dietary cholesterol and accelerates atherosclerosis.

Animals↗