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RATIONALE: Fibrocytes are progenitor cells characterized by the simultaneous expression of mesenchymal,monocyte,and hematopoietic stem cell markers. We previously documented their presence in lungs of patients with idiopathic pulmonary fibrosis. However,the mechanisms involved in their migration,subsequent homing,and local role remain unclear. Matrix metalloproteinases (MMPs) facilitate cell migration and have been implicated in the pathogenesis of pulmonary fibrosis. OBJECTIVES: To evaluate the expression and role of matrix metalloproteinases in human fibrocytes. METHODS: Fibrocytes were purified from CD14(+) monocytes and cultured for 8 days; purity of fibrocyte cultures was 95% or greater as determined by flow cytometry. Conditioned media and total RNA were collected and the expression of MMP-1,MMP-2,MMP-7,MMP-8,and MMP-9 was evaluated by real-time polymerase chain reaction. Protein synthesis was examined using a Multiplex assay,Western blot,fluorescent immunocytochemistry,and confocal microscopy. MMP-2 and MMP-9 enzymatic activities were evaluated by gelatin zymography. Migration was assessed using collagen I-coated Boyden chambers. Stromal cell-derived factor-1α and platelet-derived growth factor-B were used as chemoattractant with or without a specific MMP-8 inhibitor. MEASUREMENTS AND MAIN RESULTS: Fibrocytes showed gene and protein expression of MMP-2,MMP-9,MMP-8,and MMP-7. MMP-2 and MMP-9 enzymatic activities were also demonstrated by gelatin zymography. Likewise,we found colocalization of MMP-8 and MMP-7 with type I collagen in fibrocytes. Fibrocyte migration toward platelet-derived growth factor-B or Stromal cell-derived factor-1α in collagen I-coated Boyden chambers was significantly reduced by a specific MMP-8 inhibitor. CONCLUSIONS: Our findings reveal that fibrocytes express a variety of MMPs and that MMP-8 actively participates in the process of fibrocyte migration. View Publication

Vogt-Koyanagi-Harada (VKH) disease (and sympathetic ophthalmia) is an ocular inflammatory disease that is considered to be a cell-mediated autoimmune disease against melanocytes. The purpose of this study was to determine the Ags specific to VKH disease and to develop an animal model of VKH disease. We found that exposure of lymphocytes from patients with VKH disease to peptides (30-mer) derived from the tyrosinase family proteins led to significant proliferation of the lymphocytes. Immunization of these peptides into pigmented rats induced ocular and extraocular changes that highly resembled human VKH disease,and we suggest that an experimental VKH disease was induced in these rats. We conclude that VKH disease is an autoimmune disease against the tyrosinase family proteins. View Publication

Induced pluripotent stem cells (iPSCs) hold enormous potential for the development of personalized in vitro disease models,genomic health analyses,and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small,clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs (TiPS") retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs) with respect to morphology� View Publication

Retroviral overexpression of NF-Ya,the regulatory subunit of the transcription factor NF-Y,activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34(+) cells as a safer,more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4,a target gene of NF-Ya,using real-time polymerase chain reaction RNA and luciferase-based protein assays. After TAT-NF-Ya transduction,the proliferation of human CD34(+) cells in the presence of myeloid cytokines was increased 4-fold. Moreover,TAT-NF-Ya-treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45(+) cells recovered from the bone marrow of sublethally irradiated,transplanted NOD-Scid IL2Rγ(null) mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies and suggest that NF-Ya peptide delivery should be further evaluated as a tool for HSC/progenitors ex vivo expansion and therapy. View Publication

Femtosecond coherent anti-Stokes Raman scattering (CARS) spectroscopy offers several advantages over spontaneous Raman spectroscopy due to the inherently high sensitivity and low average power deposition in the sample. Femtosecond CARS can be implemented in a collinear pump/probe beam configuration for microspectroscopy applications and has emerged as a powerful technique for chemical imaging of biological specimens. However,one serious limitation of this approach is the presence of a high nonresonant background component that often obscures the resonant signals of interest. We report here an innovative pulse-shaping method based on Lorentzian amplitude and phase spectral modulation of a broadband femtosecond probe pulse that yields spectra with both high spectral resolution and no nonresonant background. No further mathematical analysis is needed to extract Raman spectra. The utility of the proposed method for CARS microscopy is demonstrated using a mixture of polystyrene and latex beads,as well as dry-fixed embryonic stem cells. View Publication

p400/mDomino is an ATP-dependent chromatin-remodeling protein that catalyzes the deposition of histone variant H2A.Z into nucleosomes to regulate gene expression. We previously showed that p400/mDomino is essential for embryonic development and primitive hematopoiesis. Here we generated a conditional knock-out mouse for the p400/mDomino gene and investigated the role of p400/mDomino in adult bone marrow hematopoiesis and in the cell-cycle progression of embryonic fibroblasts. The Mx1-Cre- mediated deletion of p400/mDomino resulted in an acute loss of nucleated cells in the bone marrow,including committed myeloid and erythroid cells as well as hematopoietic progenitor and stem cells. A hematopoietic colony assay revealed a drastic reduction in colony-forming activity after the deletion of p400/mDomino. Moreover,the loss of p400/mDomino in mouse embryonic fibroblasts (MEFs) resulted in strong growth inhibition. Cell-cycle analysis revealed that the mDomino-deficient MEFs exhibited a pleiotropic cell-cycle defect at the S and G(2)/M phases,and polyploid and multi-nucleated cells with micronuclei emerged. DNA microarray analysis revealed that the p400/mDomino deletion from MEFs caused the impaired expression of many cell-cycle-regulatory genes,including G(2)/M-specific genes targeted by the transcription factors FoxM1 and c-Myc. These results indicate that p400/mDomino plays a key role in cellular proliferation by controlling the expression of cell-cycle-regulatory genes. View Publication

In up to one-third of patients with acute myeloid leukemia,a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM),and this is thought to function in cancer pathogenesis. Here,we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases,caspase-6 and -8,through direct interaction with their cleaved,active forms,but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia. View Publication

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here,we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML),an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models,Cre-lox technology,and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML,while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells,such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently,myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells,resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation,and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity. View Publication

Changes in progenitor cell biology remain at the forefront of many theories of biologic aging,but there are limited studies evaluating this in humans. Aging has been associated with a progressive depletion of circulating progenitor cells,but age-related bone marrow-resident progenitor cell depletion has not been systematically determined in humans. Patients undergoing total hip replacement were consented,and bone marrow and peripheral progenitor cells were enumerated based on aldehyde dehydrogenase activity and CD34 and CD133 expression. Circulating progenitors demonstrated an age-dependent decline. In contrast,marrow-resident progenitor cell content demonstrated no age association with any progenitor cell subtype. In humans,aging is associated with depletion of circulating,but not marrow-resident,progenitors. This finding has impact on the mechanism(s) responsible for age-related changes in circulating stem cells and important implications for the use of autologous marrow for the treatment of age-related diseases. View Publication

Osteoclasts are resident cells of the bone that are primarily involved in the physiological and pathological remodeling of this tissue. Mature osteoclasts are multinucleated giant cells that are generated from the fusion of circulating precursors originating from the monocyte/macrophage lineage. During inflammatory bone conditions in vivo,de novo osteoclastogenesis is observed but it is currently unknown whether,besides increased osteoclast differentiation from undifferentiated precursors,other cell types can generate a multinucleated giant cell phenotype with bone resorbing activity. In this study,an animal model of calvaria-induced aseptic osteolysis was used to analyze possible bone resorption capabilities of dendritic cells (DCs). We determined by FACS analysis and confocal microscopy that injected GFP-labeled immature DCs were readily recruited to the site of osteolysis. Upon recruitment,the cathepsin K-positive DCs were observed in bone-resorbing pits. Additionally,chromosomal painting identified nuclei from female DCs,previously injected into a male recipient,among the nuclei of giant cells at sites of osteolysis. Finally,osteolysis was also observed upon recruitment of CD11c-GFP conventional DCs in Csf1r(-/-) mice,which exhibit a severe depletion of resident osteoclasts and tissue macrophages. Altogether,our analysis indicates that DCs may have an important role in bone resorption associated with various inflammatory diseases. View Publication

CD44+/CD24-/low tumor cells or aldehyde dehydrogenase 1 (ALDH1) positive tumor cells are considered cancer stem cells (CSCs) that possess the properties of self-renewal and tumorigenicity. However,their clinical value and significance in breast cancer remain controversial. A meta-analysis based on published studies was performed with the aim of obtaining an accurate evaluation of the association between the presence of CSCs in clinical samples and clinical outcome. A total of 12 eligible studies with 898 cases and 1,853 controls were included. CSC positive breast cancers,in particular those positive for ALDH1,were significantly associated with high histological grade,estrogen receptor (ER) negativity,progesterone receptor (PR) negativity,and human epidermal growth factor receptor type 2 (HER2) positivity. However,the presence of cancer stem cells was not associated with tumor size or nodal status. ALDH1 positive (RR = 2.83,95% CI: 2.16-3.67,P textless 0.001) and CD44+/CD24-/low tumor cells (RR = 2.32,95% CI: 1.51-3.60,P textless 0.001) were significantly associated with poor overall survival (OS). The stem cell markers are prognostic factors in breast cancer. Larger clinical studies are required to further evaluate the role of these markers in clinical practice. View Publication

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types,and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells. View Publication