text:
Telephone: 86-2984776147; Fax: +86-2983218039
Tissue-Specific Stem Cells
IFN-γ and TNF-α Synergistically Induce Mesenchymal Stem Cell Impairment and Tumorigenesis via NFκB Signaling†‡§
Version of Record online: 5 JUL 2013
DOI: 10.1002/stem.1388
Copyright © 2013 AlphaMed Press
Volume 31, Issue 7, pages 1383–1395, July 2013
Additional Information
How to Cite
Wang, L., Zhao, Y., Liu, Y., Akiyama, K., Chen, C., Qu, C., Jin, Y. and Shi, S. (2013), IFN-γ and TNF-α Synergistically Induce Mesenchymal Stem Cell Impairment and Tumorigenesis via NFκB Signaling. STEM CELLS, 31: 1383–1395. doi: 10.1002/stem.1388
- †
Author contributions: S.S., Y.J. and L.W.: conception and design, writing manuscript, and final approval of manuscript; L.W., Y.Z., Y.L., K.A., C.C. and C.Q.: collection and assembly of data; S.S., L.W., Y.Z. and Y.L.: data analysis and interpretation.
- ‡
Disclosure of potential conflicts of interest is found at the end of this article.
- §
first published online in STEM CELLS EXPRESS April 4, 2013.
Publication History
- Issue online: 5 JUL 2013
- Version of Record online: 5 JUL 2013
- Accepted manuscript online: 4 APR 2013 01:12AM EST
- Manuscript Accepted: 4 MAR 2013
- Manuscript Revised: 18 FEB 2013
- Manuscript Received: 7 DEC 2012
Funded by
- National Institute of Dental and Craniofacial Research
- National Institutes of Health
- Department of Health and Human Services. Grant Numbers: R01DE017449, R01DE019932, R01DE019413
- California Institute for Regenerative Medicine. Grant Number: RN1-00572
- National Natural Science Foundation of China. Grant Number: 81020108019
- National Basic Research Program (973 Program) of China. Grant Number: 2011CB964700
- Mesenchymal stem cells;
- Stem cell-microenvironment interactions;
- Differentiation;
- Cancer;
- Cytokines
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
An inflammatory microenvironment may cause organ degenerative diseases and malignant tumors. However, the precise mechanisms of inflammation-induced diseases are not fully understood. Here, we show that the proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) synergistically impair self-renewal and differentiation of mesenchymal stem cells (MSCs) via nuclear factor κB (NFκB)-mediated activation of mothers against decapentaplegic homolog 7 (SMAD7) in ovariectomized (OVX) mice. More interestingly, a long-term elevated levels of IFN-γ and TNF-α result in significantly increased susceptibility to malignant transformation in MSCs through NFκB-mediated upregulation of the oncogenes c-Fos and c-Myc. Depletion of either IFN-γ or TNF-α in OVX mice abolishes MSC impairment and the tendency toward malignant transformation with no NFκB-mediated oncogene activation. Systemic administration of aspirin, which significantly reduces the levels of IFN-γ and TNF-α, results in blockage of MSC deficiency and tumorigenesis by inhibition of NFκB/SMAD7 and NFκB/c-FOS and c-MYC pathways in OVX mice. In summary, this study reveals that inflammation factors, such as IFN-γ and TNF-α, synergistically induce MSC deficiency via NFκB/SMAD7 signaling and tumorigenesis via NFκB-mediated oncogene activation. STEM Cells2013;31:1383–1395
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Degenerative diseases, such as osteoporosis, usually show elevated inflammatory activity [1, 2]. Postmenopausal osteoporosis is the most common form of osteoporosis, in which estrogen deficiency-induced T-cell activation promotes osteoclastogenesis to reduce bone mineral density (BMD) [3–5]. Although antiresorptive therapies have been utilized in clinical treatment, concerns have been raised by the emergence of side effects, such as bisphosphonate-related osteonecrosis of the jaw [6, 7]. Thus, it is critical to further understand the underlying mechanisms of osteoporosis and develop novel therapeutic strategies accordingly.
Mesenchymal stem cells (MSCs) are a population of stromal progenitor cells with self-renewal and multipotent differentiation capabilities, possessing the potential to replace damaged and diseased tissues [8, 9]. Accumulating evidence shows that deficiency in MSC/osteoblast lineage consistently contributes to bone degenerative phenotypes in osteoporosis in both mice and humans [2, 10–13]. However, it remains unclear how MSCs are impaired in these immune disorder-associated diseases. If left untreated, degenerative diseases may become a risk factor for cancer occurrence [14, 15], in which chronic infections may play an important role [16]. Moreover, anti-inflammatory treatments show effectiveness in reducing the risk of cancer metastasis [17]. It was speculated that either osteosarcoma or Ewing's sarcoma may directly originate from spontaneous mutation of bone marrow-derived MSCs [18–21]. Therefore, it is possible that a long-term inflammatory environment may eventually induce MSC tumorigenesis.
It has been proved that MSCs closely interact with the host immune system. MSCs are able to inhibit proinflammatory cytokines by targeting several subsets of immune cells, including T cells, B cells, dendritic cells, and natural killer cells [22–24], and such immunoregulatory property of MSCs provides the foundation for clinical applications in treating several autoimmune diseases [2, 25]. Conversely, immune components target MSCs. For example, activated T cells can induce bone marrow MSC apoptosis via the tumor necrosis factor (TNF) receptor superfamily member 6 (FAS)/FASL and CD40/CD40L pathways, respectively [10, 26]. Lymphocytes can impair survival of implanted MSCs by secreting the proinflammatory cytokines interferon-γ (IFN-γ) and TNF-α, thereby negatively affecting MSC-mediated tissue regeneration [27, 28]. However, it remains unknown whether specific inflammatory factors, such as IFN-γ and TNF-α, affect MSC properties in an immune disorder-associated model, including ovariectomized (OVX) osteoporotic mice. Among many inflammatory cytokines, IFN-γ and TNF-α have been proved to play crucial roles in estrogen-deficient osteoporosis [29, 30]. Previous studies have shown that OVX enhances production of TNF-α by T cells, which in turn, augments macrophage colony-stimulating factor and receptor activator of nuclear factor κB (NFκB) ligand-induced osteoclastogenesis [29]. Furthermore, it has been demonstrated that OVX upregulates IFN-γ-induced class II transactivator, resulting in enhanced T-cell activation and prolonged lifespan of activated T cells [30].
Mothers against decapentaplegic homolog 7, or SMAD7, is an important inhibitory factor of the transforming growth factor β/SMAD pathway, which is one of important signaling pathways regulating osteogenic differentiation. The relationship between SMAD7 and NFκB is not clear. Although it was reported that SMAD7 inhibits nuclear translocation and transcriptional activity of the cell survival factor NFκB [31, 32], other studies demonstrated that proinflammatory cytokines, such as interleukin-1β (IL-1β) and TNF-α, upregulated SMAD7 through the activation of the NFκB pathway in fibroblasts and chondrocytes [33, 34]. Therefore, IFN-γ and TNF-α may upregulate SMAD7 via the activation of the NFκB pathway, thereby inducing the deficiency of osteogenic differentiation of MSCs. In this study, we determine that IFN-γ and TNF-α, as critical inflammatory factors, synergistically induce MSC deficiency via NFκB/SMAD7 signaling, whereas their long-term effect can result in the induction of MSC tumorigenesis by NFκB-mediated oncogene activation.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Female C3H/HeJ, C57BL/6, TNF-α knockout (KO) mice (B6;129S-Tnftm1Gkl/J) and IFN-γ KO mice (B6.129S7-Ifngtm1Ts/J) were purchased from Jackson Lab (Bar Harbor, ME, http://www.jax.org). Generation of ovariectomized (OVX) mice was performed as described previously [35] and age-matched mice receiving sham operation served as control. Female immunocompromised mice (Beige nude/nude XIDIII) were purchased from Harlan (Indianapolis, IN, http://www.harlan.com). These animal experiments were performed under institutionally approved protocols for the use of animal research (University of Southern California # 10941, 11141, and 11327).
Anti-mouse TNF-α, SMAD7, alkaline phosphatase (ALP), TNF receptor-1 (TNFR1), TNFR2, IFN-γ receptor α (IFNGRα), vimentin (VIM), pan-Cytokeratin (pan-CK), proliferating cell nuclear antigen (PCNA), c-FOS, and c-MYC antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA, http://www.scbt.com); IFN-γ antibody was purchased from Abbiotec (San Diego, CA, http://www.abbiotec.com); phosphorylated nuclear factor κB (p-NFκB), NFκBp65, p-NFκBp50, NFκBp50, phosphorylated nuclear factor κ B inhibitor (p-IκB), IκBα, p-SMAD1, SMAD1, runt-related transcription factor 2 (RUNX2), and signal transducers and activators of transcription factor 1 (STAT-1) were purchased from Cell Signaling (Danvers, MA, http://www.cellsignal. com). Anti-β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com).
Measurement of TNF-α and IFN-γ in Blood Serum, Bone Marrow, Derma, and Fat
Peripheral blood was collected from retro-orbital venous plexus, and IFN-γ and TNF-α enzyme-linked immunosorbent assay (ELISA) (BioLegend, San Diego, CA, http://www.bio legend.com) was performed to detect in blood serum according to their manufactures' instructions. Total protein from bone marrow, derma, and subcutaneous fat was extracted using M-PER mammalian protein extraction reagent (Thermo, Rockford, IL, http://www.thermoscientific.com), and IFN-γ and TNF-α were measured by ELISA, respectively.
Isolation of Mouse MSCs
For isolation of bone marrow MSCs, bone marrow cells were flushed out from bone cavity of femurs and tibias of mice with 2% heat-inactivated fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, http://www.equitech-bio.com) in phosphate-buffered saline (PBS). For isolation of dermal MSCs and subcutaneous fat MSCs, dermis and subcutaneous fat were carefully separated from the full skin under a dissecting microscope, cut into small pieces, and digested with 1.5 mg/ml collagenase type I (Worthington, Lakewood, NJ, http://www.worthington-biochem.com) and Dispase (Roche, Indianapolis, IN, http://www.roche-applied-science.com) in PBS 2 mg/ml for 1 hour at 37°C to release the individual cells. A single-cell suspension of all nucleated cells was obtained by passing all bone marrow cells through a 70-μm cell strainer (BD Bioscience, San Jose, CA, http://www.bdbiosciences. com), respectively. All the single cells were seeded into 100-mm culture dishes (Corning, Corning, NY, http://www. corning.com) and initially incubated for 48 hour at 37°C and 5% CO2. To eliminate the nonadherent cells, the cultures were washed with PBS twice on the second day. The attached cells were cultured for 16 d with alpha minimum essential medium (α-MEM, Invitrogen, Grand Island, NY, http://www.invitrogen.com) supplemented with 20% FBS, 2 mM L-glutamine (Invitrogen), 55 μM 2-mercaptoethanol (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). To confirm mesenchymal stem cell character, flow cytometric analysis was used to show that these MSCs were positive for CD73, CD90, Sca-1 and SSEA-4 and negative for CD11b, CD31, CD34, and CD45.
Population-Doubling Assay
Population doubling (PD) for each passage was determined using the formula PD≈log2[Nc/No], where No is the original cell population and Nc is the number of cells at confluence, and by adding the PD from each passage together to obtain the PD values. The PD assay for mouse MSCs began with passage 1.
Osteogenic Differentiation of MSCs
For osteogenic induction in vitro, mouse MSCs were seeded on 35-mm dishes (Corning) and cultured in the growth medium until the cells reached confluence. To induce osteogenic differentiation, the medium was changed to an osteogenic medium consisting of L-ascorbic acid phosphate and β-glycerophosphate. After 2 weeks of osteogenic induction, total protein was extracted and analyzed for osteogenic markers by Western blot. After 4–6 weeks, calcium deposits formed by osteoblasts on the dishes were detected by staining with 2% Alizarin Red (Sigma-Aldrich).
Histological and Histomorphometric Analysis
Femurs and skins were fixed with 4% paraformaldehyde, followed by decalcification with 10% EDTA (pH 8.0) for femurs, and embedded in paraffin. For histological and histomorphometric analysis, sections were deparaffinized and stained with hematoxylin and eosin (HE), followed by trabecular percentage and derma thickness calculation using ImageJ software.
Immunohistochemistry Staining
For the detection of TNF-α, IFN-γ, VIM, pan-CK, PCNA, p-NFκBp65, c-FOS, and c-MYC, the sections were blocked with serum matched to secondary antibodies, incubated with the first antibodies for 30 min at room temperature, and stained using VECTASTAIN Elite ABC Kit (Vector, Burlingame, CA, http://www.vectorlabs.com) and DAB Kit (Invitrogen), according to the manufacturers' instructions.
Administration of IFN-γ and TNF-α Neutralizing Antibodies in OVX Mice
To chronically deplete IFN-γ and TNF-α, at 1-day before OVX, 12-week-old female C3H mice were injected i.p. with 250 μg of IFN-γ and TNF-α neutralizing antibodies (BioLegend) in 0.5 ml sterile saline every 3 days until 21 days post-OVX, respectively. In control groups, 250 μg isotype control antibodies in 0.5 ml sterile saline were identically administered.
Administration of IFN-γ and TNF-α in Nude Mice
To mimic IFN-γ- and TNF-α-induced MSC deficiency in an in vivo model, 8-week-old female nude mice were injected with 4 μg recombinant mouse IFN-γ (BioLegend) and 1 μg recombinant mouse TNF-α (BioLegend), either alone or in combination, in 0.2 ml sterile saline via tail vein every 3 days for 21 days, respectively.
Inhibition of Osteogenic Differentiation by IFN-γ and TNF-α Treatment
Half a million MSCs were seeded onto each 6-well culture dish (Corning) with or without treatment using IκB kinase α (IKKα), SMAD7, TNFR1, or IFNGRα small interfering RNA (siRNA) (Santa Cruz) according to the manufacturer's instruction. IFN-γ and TNF-α, either alone or incombination, were used to treat the MSCs with normal growth media or osteogenic media for 14 days. Total protein was extracted for detection of NFκB pathway and osteogenic markers using Western blot. For some experiments, the culture plates were stained with 2% toluidine blue O and 2% paraformaldehyde to evaluate cell survival.
Western Blot Analysis
Total protein was extracted using M-PER mammalian protein extraction reagent (Thermo). Nuclear protein was obtained using NE-PER nuclear and cytoplasmic extraction reagent (Thermo). Protein was applied and separated on 4–12% NuPAGE gel (Invitrogen) and transferred to ImmobilonTM-P membranes (EMD Millipore, Billerica, MA, http://www.emd millipore.com). After blocking with 5% nonfat dry milk for 1 hour, the membranes were incubated with the primary antibodies (1:200–1,000 dilution) at 4°C overnight. Horseradish peroxidase-conjugated IgG (Santa Cruz) was used to treat the membranes for 1 hour and subsequently treated with a chemiluminescent substrate (Thermo). The bands were detected on BIOMAX MR films (Kodak, Rochester, NY, http://www. kodak.com). Each membrane was also stripped using a stripping buffer (Thermo) and reprobed with anti-β-Actin to determine the loading amount.
Sarcomagenesis of BMMSCs in MCA-Treated Mice
To determine the susceptibility to tumorigenesis of bone marrow MSCs (BMMSCs) in vivo, we locally applied 3-methycholanthrene (MCA) to the bone marrow of tibias as a carcinogen to induce osteosarcoma according to the method described in previous studies with modifications [36, 37]. One month after OVX or sham operation, a small hole was created in the metaphysis of right tibia using a dental bur after anesthesia, and a single dose of 0.5 mg MCA in 10 μl of dimethyl sulfoxide (DMSO) was injected into the bone marrow of tibia, followed by sealing using bone wax. Control mice received identical procedures except that DMSO without MCA was implanted into tibias. MCA-treated mice were closely observed for tumor occurrence for up to 4 months. MCA-treated tibias were harvested, demineralized with 10% EDTA, and embedded for HE, Trichrome staining, and immunostainings for VIM, pan-CK, and PCNA.
MSC Spontaneous Malignant Transformation Assay
To evaluate the effects of inflammatory microenvironment to MSC malignant transformation in vitro, we treated mouse BMMSCs with long-term IFN-γ (50 ng/ml) and TNF-α (5 ng/ml), with or without treatment of NFκB inhibitor BAY 11-7082 (1 μM, Sigma-Aldrich), and continuously passaged to gradually acquire unlimited population doublings as previously described [18]. Giemsa staining was performed to monitor the cell shape.
Transplantation and Injection of BMMSCs
To assess tumorigenesis and migration potential, 2 × 106 and 1 × 106 of BMMSCs were injected into 8-week-old nude mice subcutaneously and intravenously through tail vein, respectively. Transplants and lungs were harvested 8 weeks after transplantation, and the paraffin-embedded sections were stained with HE, Trichrome, or incubated with VIM, pan-CK, and PCNA antibodies (Santa Cruz) for immunohistochemistry analysis.
Semiquantitative Real-Time PCR
Total RNA was isolated from the cultures using SV total RNA isolation kit (Promega, Madison, WI, http://www. promega.com) following the manufacturer's protocols. The cDNA was synthesized from 100 ng of total RNA using Superscript III (Invitrogen). Polymerase chain reaction (PCR) was performed on a CFX 96 real-time PCR thermocycler (BioRad, Hercules, CA, http://www.bio-rad.com) using gene-specific primers and Cybergreen supermix (BioRad). Primer pairs are as follows: mouse c-Fos, forward primer (AAACCGCATGGAGTGTGTTGTTCC) and reverse primer (TCAGACCACCTCGACAATGCATGA); mouse c-Myc, forward primer (GCCACGTCTCCACACATCAG) and reverse primer (TCTTGGCAGCAGGATAGTCCTT). The conditions for amplification were as follows: 94°C for 5 min, followed by 40 cycles of 94°C for 20 s; 60°C for 20 s; and 72°C for 25 s. Data from the real-time PCR reactions were analyzed using CFX Manager Software, Version 2 (Bio-Rad). Relative changes in mRNA expression were analyzed using the 2–ΔΔCt method, with Gapdh serving as an internal reference to correct for differences in RNA extraction or reverse transcription efficiencies. These standardized data were used to calculate fold-differences in gene expression. All real-time PCR amplifications were performed in triplicate.
Aspirin Administration in OVX Mice
To determine the effects of aspirin to prevent MSC tumorigenesis and MSC deficiency in vivo, mice were fed with aspirin (1 mg/ml, Sigma-Aldrich) dissolved in water or placebo for 2 months in OVX mice and 4 months in MCA-treated OVX mice. BMMSCs and DMSCs were harvested for PD and osteogenic differentiation analysis.
SPSS 13.0 was used to perform statistical analysis. Significance was assessed by independent two-tailed Student's t test or analysis of variance. The p < 0.05 were considered significant.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Elevated Levels of IFN-γ and TNF-α Synergistically Impair MSCs in OVX Mice
Of many immune disorder-associated degenerative diseases, postmenopausal osteoporosis is an important example. It features overactivation of proinflammatory cytokine-producing T cells and decreased osteogenic differentiation capabilities in bone marrow MSCs (BMMSCs), resulting in reduced bone mineral density (BMD) [2–5, 10]. IFN-γ and TNF-α have been proved to play crucial roles in estrogen-deficient osteoporosis [29, 30]. Blockage of either IFN-γ or TNF-α can abolish OVX-induced osteoporotic phenotype [3, 5]. Moreover, OVX enhances T-cell activation and prolonged lifespan of active T cells through IFN-γ-induced class II transactivator [30]. Our recent study also showed that IFN-γ and TNF-α synergistically impaired implanted MSCs in vivo [28]. Based on these observations, we asked whether IFN-γ and TNF-α would affect MSC properties in OVX-induced osteoporotic mice. We hypothesized that IFN-γ and TNF-α, as two key representatives in the inflammatory microenvironment, might synergistically play an important role in inducing MSC deficiency in OVX-induced osteoporotic mice. Concentrations of both IFN-γ and TNF-α in serum, bone marrow and derma, but not in subcutaneous fat, were markedly increased in OVX mice when compared with sham-operated mice by ELISA and immunohistochemical staining analysis (Fig. 1A; Supporting Information Fig. S1). BMMSCs derived from OVX mice showed markedly decreased population doublings when compared with sham mouse-derived BMMSCs, indicating that BMMSC self-renewal capability was impaired in OVX mice (Fig. 1B). Importantly, osteogenic differentiation of BMMSCs was also impaired in OVX mice, as indicated by Alizarin red staining to show markedly decreased mineralized nodule formation after induction of osteogenic media (Fig. 1C). Because derma and subcutaneous fat, as neighboring tissues, showed different alteration in the levels of IFN-γ and TNF-α following OVX procedure (Fig. 1A), we speculate that OVX may affect derma and subcutaneous fat MSCs differently from bone marrow MSCs. Although OCT-4 is not a specific marker for MSCs, it has been proved to be an important marker to indicate that MSCs possess self-renewal capacity and can retain their undifferentiated status [38]. Therefore, we used OCT-4 as a marker of functional stem cell assurance for MSCs. We found that the derma, but not the fat, from OVX mice harbored markedly less OCT-4-positive MSCs (Fig. 1D), contributing to a markedly decreased collagen deposition in the derma as shown by Trichrome staining, when compared with sham-operated mice (Fig. 1E). To further confirm decreased MSC number in OVX mice, we performed histoimmunostaining to show markedly reduced number of dermal MSCs, which express the MSC markers CD105, CD44 and CD73 in OVX mice (Supporting Information Fig. S2). Moreover, OVX-derived dermal MSCs (DMSCs), but not fat MSCs (FMSCs), showed markedly decreased population doublings (Fig. 1F) and osteogenic differentiation (Fig. 1G), which served as a representative to determine the differentiation capabilities of MSCs in the experiments thereafter. Consequently, OVX mice showed markedly deteriorated trabecular bone structures and reduced dermal thickness when compared with sham-operated mice (Supporting Information Fig. S3A, S3B).
We next asked whether elevated levels of IFN-γ and TNF-α would synergistically cause MSC deficiency. To clarify this, we first systemically administered either IFN-γ or TNF-α neutralizing antibody to OVX mice to test whether depletion of IFN-γ or TNF-α could prevent OVX-induced MSC deficiency. Indeed, administration of IFN-γ neutralizing antibody was able to markedly reduce IFN-γ level in the serum of OVX mice, without affecting TNF-α level; similarly, administration of TNF-α neutralizing antibody could markedly reduce the serum TNF-α level in OVX mice, without changing IFN-γ level (data not shown). Interestingly, histological analysis showed that either IFN-γ or TNF-α neutralizing antibody was able to markedly inhibit bone loss in OVX mice (Fig. 2A). MSC characterization showed that either IFN-γ or TNF-α neutralizing antibody treatment in vivo markedly improved population doublings and osteogenic differentiation in OVX-derived BMMSCs (Fig. 2B). Also, either IFN-γ or TNF-α neutralizing antibody was able to prevent derma atrophy in OVX mice (Fig. 2C) and improve population doublings and differentiation capabilities in OVX-derived DMSCs (Fig. 2D). To further dissect whether IFN-γ and TNF-α synergistically cause MSC deficiency in OVX mice, we confirmed that OVX was not able to induce BMD loss in either IFN-γ KO or TNF-α KO mice (Fig. 2E, 2F). Importantly, population doublings and differentiation capabilities were not decreased in BMMSCs or DMSCs in IFN-γ KO or TNF-α KO mice after OVX (Fig. 2E, 2F). To further confirm the synergistic effect of IFN-γ and TNF-α in vivo, we systemically infused IFN-γ and TNF-α, either alone or in combination, to immunocompromised mice via tail vein. Interestingly, infusion of IFN-γ and TNF-α in combination, but not separately, caused a dramatic reduction in population doublings and differentiation capabilities of both BMMSCs and DMSCs (Fig. 3A–3D), resulting in both osteopenia phenotype and dermal atrophy with poor collagen deposition as shown by Trichrome staining (Fig. 3E–3G; Supporting Information Fig. S4A, S4B). Furthermore, when IFN-γ and TNF-α, either alone or in combination, were subcutaneously transplanted with hydrogel as a controlled release system, DMSC population doublings and differentiation capabilities were reduced by IFN-γ and TNF-α in combination, but not separately (Supporting Information Fig. S5A, S5B), thereby causing derma atrophy (Supporting Information Fig. S5C). These in vivo data suggest that elevated levels of IFN-γ and TNF-α synergistically, but not independently, cause MSC deficiency.
IFN-γ and TNF-α Synergistically Induce NFκB/SMAD7 Pathway Activation, Causing Differentiation Deficiency in MSCs
To examine the mechanism by which IFN-γ and TNF-α synergistically induce impairment of MSCs, we demonstrated that BMMSCs and DMSCs derived from OVX mice showed the upregulation of p-NFκB, p-IκB, and inhibitory SMAD7, but the downregulation of osteogenic genes, including phosphorylated SMAD1 (p-SMAD1), RUNX2, and ALP (Fig. 4A; Supporting Information Fig. S6A). To mimic in vivo microenvironment in OVX bone marrow, we treated BMMSCs with IFN-γ and TNF-α in different concentrations, either alone or in combination, and found that TNF-α-induced NFκBp65 activation in a dose-dependent manner with a maximal activation at concentrations of 5 and 20 ng/ml (Supporting Information Fig. S6B), whereas the combination of IFN-γ (50 ng/ml) and TNF-α (5 ng/ml) induced more significant upregulation of NFκBp65 signaling (Fig. 4B). Interestingly, IFN-γ and TNF-α in combination, but not separately, caused a significant reduction of mineralized nodule formation in BMMSCs (Fig. 4C) and DMSCs cultured in osteogenic media (Supporting Information Fig. S6C, S6D), without dramatically affecting MSC survival (Fig. 4E). Furthermore, Western blot showed that IFN-γ/TNF-α-induced NFκB activation upregulated inhibitory SMAD7, but downregulated osteogenic genes, including p-SMAD1, RUNX2, and ALP (Fig. 4F). When NFκB expression was knocked down by IKKα siRNA, IFN-γ/TNF-α-induced upregulation of SMAD7 and downregulation of p-SMAD1, RUNX2, and ALP were abolished, leading to the rescue of IFN-γ/TNF-α-induced reduction of mineralized nodule formation in BMMSCs (Fig. 4G, 4H) and DMSCs (Supporting Information Fig. S6E, S6F). Under osteogenic induction, IFN-γ and TNF-α treatment in combination was able to dramatically upregulate IFN-γ receptor IFNGRα and TNF-α receptor TNFR1, but not TNFR2, when compared with IFN-γ and TNF-α treatment alone (Fig. 4D). Combination knockdown of IFNGRα and TNFR1 resulted in marked inhibition of IFN-γ-/TNF-α-induced activation of NFκB pathway when compared with knockdown of TNFR1 alone, suggesting that IFN-γ activates TNF-α-induced NFκB activation via IFNGRα (Supporting Information Fig. S7A). Moreover, IFN-γ, either alone or in combination with TNF-α, was able to markedly upregulate p-STAT-1 (Fig. 4D), whereas knockdown of STAT1 dramatically reduced the TNF-α-/IFN-γ-induced activation of NFκB pathway, suggesting that IFN-γ/IFNGRα pathway enhances TNF-α-triggered NFκB activation via STAT-1 activation (Supporting Information Fig. S7B). We confirmed the efficiency of IKKα, IFNGRα, TNFR1, STAT-1 and SMAD7 knockdown by their spe-cific siRNAs using Western blot analysis (Supporting Information Fig. S7C, S7D). Additionally, knockdown of SMAD7 expression by siRNA assay resulted in rescuing TNF-α-/IFN-γ-induced reduction of mineralized nodule formation and downregulation of p-SMAD1, RUNX2, and ALP in BMMSCs and DMSCs, without affecting p-IκB and p-NFκB (Supporting Information Fig. S8A–S8D). These data suggest that SMAD7 acts as a downstream target of NFκB signaling in TNF-α-/IFN-γ-induced MSC osteogenic deficiency.
To further confirm the role of the NFκB pathway in TNF-α-/IFN-γ-induced MSC deficiency, we showed that NFκB inhibitor Bay 11-7082 treatment was able to rescue osteogenic differentiation of OVX-derived BMMSCs by downregulating SMAD7 and upregulating p-SMAD1, RUNX2, and ALP (Fig. 4I, 4J). To confirm the synergistic effects of IFN-γ and TNF-α that resulted in MSC deficiency in vivo, we demonstrated that OVX failed to induce MSC NFκB activation in IFN-γ and TNF-α KO mice, respectively (Fig. 4K, 4L). Moreover, either IFN-γ or TNF-α neutralizing antibody treatment in vivo was sufficient to downregulate NFκB pathway in BMMSCs to the level observed in the sham-operated group (Fig. 4M). These in vivo results confirm that IFN-γ and TNF-α work synergistically to induce osteogenic deficiency in MSCs via NFκB pathway.
IFN-γ and TNF-α Increase the Tendency Toward MSC Malignant Transformation via NFκB-Mediated Upregulation of Oncogenes c-Fos and c-Myc
Given the facts that degenerative diseases may be a risk factor for cancer occurrence [14] and that NFκB activation may upregulate oncogenes c-Fos and c-Myc expression [39], we next asked whether IFN-γ-/TNF-α-induced NFκB activation could increase the tendency toward MSC malignant transformation. When we extended the observation time to 1-year post-OVX, the aging OVX mice showed a markedly increased tumor incidence when compared with aging sham-operated mice (Fig. 5A). Interestingly, ELISA showed that both IFN-γ and TNF-α concentration in serum remained markedly higher in OVX mice when compared with sham-operated mice (Fig. 5B), suggesting that long-term overexposure of MSCs to IFN-γ and TNF-α may be associated with higher susceptibility to tumorigenesis in OVX mice. To confirm that such higher susceptibility of tumorigenesis involves MSCs, we locally applied 3-methycholanthrene (MCA) as a carcinogen to induce osteosarcoma [36, 40], and found that the MCA-treated OVX mice showed a higher incidence of sarcoma with earlier sarcoma occurrence in femurs when compared with sham-operated mice (Fig. 5C). In tumors that occurred in MCA-treated OVX mice, but not sham-operated mice, histological analysis showed the production of osteoid by atypical tumor cells with high mitotic activity and abundant neovascularization, as well as osteogenic differentiation of the osteosarcoma-like tumor cells, as indicated by Trichrome staining (Fig. 5D). Immunohistochemical costaining showed that CD73 was highly overlapped with VIM or PCNA (Fig. 5E), confirming that the highly proliferating tumor cells mainly originated from mesenchymal cells, while excluding the possibility of epithelial cell origin, as indicated by negative staining of pan-CK (Supporting Information Fig. S9A). Furthermore, MCA-treated OVX mice showed a markedly lower incidence of sarcoma with delayed sarcoma occurrence in IFN-γ KO and TNF-α KO mice, respectively, when compared with the sham-operated mice (Fig. 5F), suggesting again that IFN-γ and TNF-α work synergistically to, in this case, induce sarcomagenesis.
These findings provided a strong rationale for further exploring the mechanisms by which long-term IFN-γ/TNF-α overexposure could induce MSC malignant transformation. To achieve this, we used an established spontaneous malignant transformation assay [18] by continuously passaging mouse BMMSCs to gradually acquire unlimited population doublings. When treated with long-term IFN-γ/TNF-α in vitro, the BMMSCs showed a markedly quicker increase in population doublings after passage 10, whereas NFκB inhibitor was able to delay such increase in population doublings (Fig. 6A). At passage 12, IFN-γ-/TNF-α-treated BMMSCs showed markedly smaller morphology with frequent abnormal mitosis and the generation of sarcoma-like tumors on s.c. inoculation, which, when infused to nude mice via tail vein settled in the lungs with in situ tumorigenesis (Fig. 6B). In contrast, neither NFκB inhibitor-treated mice nor control mice showed any change in cell morphology or generated any tumor on s.c. inoculation or infusion into nude mice (Fig. 6B). Immunohistochemical staining of the tumors derived from s.c. inoculation of IFN-γ-/TNF-α-treated BMMSCs revealed that the highly proliferating tumor cells originated from mesenchymal cells, as indicated by high expressions of VIM and PCNA and negative staining of pan-CK (Supporting Information Fig. S9B).
To further dissect the molecular mechanism of such IFN-γ-/TNF-α-induced MSC tumorigenesis, we demonstrated that the expression of c-MYC and c-FOS, was markedly elevated in long-term IFN-γ-/TNF-α-treated BMMSCs when compared with control BMMSCs (Fig. 6C), but that knockdown of NFκB by IKKα siRNA assay was able to abolish the elevation in c-MYC and c-FOS (Fig. 6C). To confirm this result in vivo, we showed that c-Myc and c-Fos were markedly elevated in BMMSCs derived from aging OVX mice when compared with sham-operated ones using real-time PCR (Fig. 6D), which was confirmed by Western blot and immunohistochemistry showing that c-MYC and c-FOS expression was enhanced along with NFκB activation in aging OVX mice when compared with sham-operated mice (Fig. 6E, Supporting Information Fig. S9C).
Aspirin Blocks OVX-Induced MSC Deficiency and Tumorigenesis by Inhibiting IFN-γ and TNF-α
Next, we investigated whether blocking the levels of IFN-γ and TNF-α by pharmacological approach could rescue MSC deficiency and tumorigenesis in vivo. As aspirin has been reported to inhibit the function of IFN-γ and TNF-α [28, 41], we examined the effect of systemic aspirin treatment in OVX mice which had undergone MCA injections in their tibias. Interestingly, aspirin treatment dramatically reduced the concentrations of IFN-γ and TNF-α in both serum and bone marrow, resulting in a decreased and delayed osteosarcoma occurrence following MCA treatment (Fig. 7A, 7B), with marked inhibition of NFκB activity in BMMSCs, as shown by Western blot analysis (Fig. 7C). The data suggest that aspirin treatment may decrease the tendency toward MSC malignant transformation by inhibiting IFN-γ-/TNF-α-induced NFkB activation.
To determine the efficacy of aspirin administration to rescue IFN-γ-/TNF-α-induced MSC deficiency in organ degenerative diseases, we systemically applied aspirin to OVX mice and found that aspirin could markedly improve BMD and rescue the deteriorated trabecular bone structures and derma atrophy in OVX mice (Fig. 7D; Supporting Information Fig. S10A, S10B), at least partly by reducing the concentrations of IFN-γ and TNF-α in serum, bone marrow and derma (Fig. 7E). Importantly, in vivo aspirin administration was able to markedly improve population doublings and differentiation capacities of both BMMSCs and DMSCs in OVX mice (Fig. 7F–7H), suggesting that aspirin treatment may provide an ideal MSC-based therapy for OVX mice by preventing MSC deficiency which is induced by medium-term IFN-γ and TNF-α overexposure.
In summary, we have uncovered a previously unrecognized mechanism whereby elevated levels of IFN-γ and TNF-α synergistically, but not independently, cause MSC deficiency via NFκB-mediated activation of SMAD7 in OVX mice, whereas long-term elevated levels of IFN-γ and TNF-α synergistically result in increased tendency toward MSC malignant transformation through NFκB-mediated upregulation of the oncogenes c-Fos and c-Myc (Supporting Information Fig. S11). Thus, as a therapeutic regimen, systemic administration of aspirin provides a promising approach in rescuing MSC deficiency in organ degenerative diseases, as well as preventing MSC tumorigenesis through reducing the levels of IFN-γ and TNF-α.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
It has been well documented that estrogen deficiency increases levels of IFN-γ and TNF-α thereby enhancing osteoclast activity in OVX mice [3–5]. However, it has been unclear whether elevated levels of IFN-γ and TNF-α contribute to the impairment of MSCs/osteoblasts in OVX mice or osteoporotic humans [2, 10–13]. In this study, we found that elevated levels of IFN-γ and TNF-α synergistically induce osteogenic deficiency of MSCs through NFκB/SMAD7, whereas long-term overexposure of IFN-γ and TNF-α can induce malignant transformation of MSCs.
TNF-α is widely accepted as a proinflammatory cytokine, serving as a prerequisite in several models of inflammatory and autoimmune diseases. However, IFN-γ is believed to possess both proinflammatory and anti-inflammatory properties, such as inhibiting IL-1 production, and, as such, it also plays a complex role in inflammation and autoimmune diseases [42]. The respective effects of IFN-γ and TNF-α in osteogenic differentiation of MSCs have remained controversial [28, 43–47]. The discrepancies in the effects of IFN-γ and TNF-α among these studies may be attributed to the different doses of IFN-γ and TNF-α, treatment, variations between the microenvironments and MSC growth status. Nonetheless, IFN-γ and TNF-α do not present independently in vivo; instead, they are simultaneously secreted by activated Th1 and other immune cells and cooperatively create proinflammatory microenvironments. Indeed, our data demonstrated that high levels of IFN-γ and TNF-α synergistically play a crucial role in causing MSC deficiency in OVX mice, thereby causing the phenotypes of osteoporotic bone or derma atrophy in OVX mice. Blockage of either IFN-γ or TNF-α with their neutralizing antibodies in vivo is sufficient to effectively rescue MSC deficiency. Furthermore, OVX is not able to induce MSC deficiency in IFN-γ and TNF-α KO mice respectively, suggesting that IFN-γ and TNF-α act together, not independently, to induce MSC deficiency in the OVX model. As OVX can induce elevation of multiple inflammatory cytokines, it is possible that other cytokines, with the exception of IFN-γ and TNF-α, may contribute to OVX-induced MSC impairment in OVX mice. For example, IL-1 has been proved to enhance osteoclastogenesis in OVX mice [48]. Thus, further study is required to clarify the role of other inflammatory cytokines in OVX-induced osteoporosis. In this study, we demonstrate that IFN-γ and TNF-α play a crucial role in synergistically inducing MSC deficiency in OVX mice. Given the fact that MSCs derived from derma and subcutaneous fat have different changes in population doublings and osteogenic differentiation after OVX, the effects of IFN-γ and TNF-α in inducing MSC deficiency may be associated with tissue levels of IFN-γ and TNF-α.
It has been demonstrated that IFN-γ and TNF-α may activate STAT-1 and NFκB pathways, thereby interacting with their common basal transcription complexes to affect coactivator recruitment in fibroblasts and asthmatic airway smooth muscle cells, respectively [49, 50]. IFN-γ and TNF-α have also been reported to synergistically activate STAT-1/IFN regulatory factor-1 and c-Jun NH2-terminal kinase (JNK) pathways to induce apoptosis of pancreatic β-cells in autoimmune diabetes [51, 52]. However, it is unclear how overexposure of stem cells to the synergistically activity of IFN-γ and TNF-α affects their behavior during the course of degenerative diseases. In this study, we demonstrated that IFN-γ binds its receptor IFNGR1 to strongly enhance TNF-α/TNFR1-upregulated NFκB, which, in turn, mediates inhibitory SMAD7 to downregulate the osteogenic master gene Runx2 [53] and, hence, induce osteogenic deficiency in MSCs, both in vivo and in vitro. In our previous studies, we proved that TNF-α converts the signaling of the IFN-γ-activated, nonapoptotic form of FAS in BMMSCs to a caspases-associated proapoptotic cascade, resulting in the apoptosis of implanted BMMSCs [28]. Such difference in the final fates of MSCs may be caused by high levels of IFN-γ and TNF-α produced by more intensive local infiltration of Th1 cells in tissue-engineered transplants when compared with those in a degenerative disease model, such as OVX mice. The synergistic dose-dependent effects of IFN-γ and TNF-α indicate that proinflammatory microenvironments in degenerative diseases and tissue engineering are hierarchical and complex for the alteration of MSCs.
It has been suggested that TNF-α may drive malignant tumor progression and that NFκB signaling may be involved in tumor development [16, 54, 55]. However, it has been unknown whether long-term overexposure of synergistically active IFN-γ and TNF-α could cause malignant transformation of MSCs. In this study, we extended the period of IFN-γ and TNF-α overexposure and found that the ultimate effect was upregulation of the oncogenes c-Fos and c-Myc via NFκB signaling, thereby causing increased tendency toward MSC tumorigenesis, both in vitro and in vivo. Furthermore, carcinogen-treated OVX IFN-γ and TNF-α KO mice showed a markedly lower incidence of sarcoma with delayed sarcoma occurrence, indicating that IFN-γ and TNF-α must work together, not independently, to upregulate NFκB-mediated oncogenes c-Fos and c-Myc. Thus, it is plausible that both organ degenerative diseases and malignant tumor might be "stem cell diseases", in which IFN-γ and TNF-α overexposure plays a dynamic role in inducing MSC deficiency and MSC tumorigenesis through activation of NFκB-mediated SMAD7 and the oncogenes c-Fos/c-Myc, respectively.
As a widely used anti-inflammatory drug, aspirin is able to inhibit osteoclastogenesis and improve osteogenesis in OVX mice by inhibiting cyclooxygenases and upregulating telomerase in BMMSCs [10, 56]. As aspirin can block Th1 cell development [57] and reduce TNF-α- and IFN-γ-producing cells in implanted tissue-engineered bones [28], we hypothesized that aspirin should be able to block TNF-α and IFN-γ in OVX mice and therefore rescue MSC deficiency and MSC tumorigenesis. Indeed, we demonstrated that systemic administration of aspirin was able to decrease concentrations of TNF-α and IFN-γ, both in serum and bone marrow, and prevent MSC deficiency and carcinogen-induced MSC tumorigenesis in OVX mice. Therefore, aspirin may be a promising drug to treat estrogen deficiency-induced MSC deficiency and reduce the risk of tumor occurrence. However, cautious evaluation is needed before these findings in mice would accurately translate to humans. On the one hand, although aspirin was shown to be associated with higher BMD at multiple skeletal sites in a cohort study of men and women [58], Bauer et al. [59] only observed a modest beneficial effect on BMD in another cohort study of postmenopausal women. On the other hand, although daily aspirin has been proved to decrease the risk of adenocarcinoma metastasis in a randomized controlled trial [17], a recent study indicated that regular administration of aspirin and other nonsteroidal anti-inflammatory drugs was associated with a slightly higher incidence of follicular lymphoma incidence [60]. Thus, the daily use of aspirin or nonsteroidal anti-inflammatory drugs and the risk of cancer in human should be investigated carefully using large cohorts of patients. Also, large cohort studies are required to determine the effects of aspirin in treating post-menopausal osteoporosis and other organ degenerative diseases. Moreover, it is known that MSCs possess such immunomodulatory properties as immunosuppression [61]. Therefore, it will be interesting to examine whether host MSCs show any immunosuppressive effects, such as inhibition of activated T cells and NK cells, thereby serving as a self-protecting mechanism against potential autoimmune reactions by the host immune system.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
In summary, this study demonstrated that TNF-α and IFN-γ, as two important representative inflammatory factors, play a crucial role in synergistically inducing MSC deficiency and eventual MSC tumorigenesis via the NFκB pathway in OVX-induced osteoporotic mice. Anti-inflammatory treatment, such as aspirin administration, prevents MSC deficiency and malignant transformation in OVX mice.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
This work was supported by grants from National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services (R01DE017449, R01DE019932, and R01DE019413; to S.S.), grant from California Institute for Regenerative Medicine (RN1-00572; to S.S.), grant from the National Natural Science Foundation of China (No. 81020108019; to Y.J. and S.S.), and grant from the National Basic Research Program (973 Program) of China (No. 2011CB964700; to Y.J.).
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
The authors declare that they have no competing interests.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
- 1The multiple faces of autoimmune-mediated bone loss. Nat Rev Endocrinol 2010; 6: 698–706., .
- 2Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans. Stem Cells 2009; 27: 1421–1432., , et al.
- 3IFN-gamma stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J Clin Invest 2007; 117: 122–132., , et al.
- 4Estrogen deficiency and bone loss: an inflammatory tale. J Clin Invest 2006; 116: 1186–1194., .
- 5Up-regulation of TNF-producing T cells in the bone marrow: a key mechanism by which estrogen deficiency induces bone loss in vivo. Proc Natl Acad Sci U S A 2001; 98: 13960–13965., , et al.
- 6Osteoporosis: now and the future. Lancet 2011; 377: 1276–1287., , .
- 7Cell-based immunotherapy with mesenchymal stem cells cures bisphosphonate-related osteonecrosis of the jaw-like disease in mice. J Bone Miner Res 2010; 25: 1668–1679., , et al.
- 8The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 1970; 3: 393–403., , .
- 9Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 2003; 18: 696–704., .
- 10Pharmacologic stem cell based intervention as a new approach to osteoporosis treatment in rodents. Plos One 2008; 3: e2615., , et al.
- 11Mouse mandible contains distinctive mesenchymal stem cells. J Dent Res 2011; 90: 317–324., , et al.
- 12Inhibition of osteoblastic bone formation by nuclear factor-kappaB. Nat Med 2009; 15: 682–689., , et al.
- 13The transcriptional profile of mesenchymal stem cell populations in primary osteoporosis is distinct and shows overexpression of osteogenic inhibitors. Plos One 2012; 7: e45142., , et al.
- 14
- 15Gender-related hormonal risk factors for oral cancer. Pathol Oncol Res 2007; 13: 195–202..
- 16Infections as a major preventable cause of human cancer. J Intern Med 2000; 248: 171–183., , .
- 17Effect of daily aspirin on risk of cancer metastasis: a study of incident cancers during randomised controlled trials. Lancet 2012; 379: 1591–1601., , et al.
- 18Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 2006; 24: 1095–1103., , et al.
- 19Osteosarcoma originates from mesenchymal stem cells in consequence of aneuploidization and genomic loss of Cdkn2. J Pathol 2009; 219: 294–305., , et al.
- 20Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal progenitor cells. Cancer Res 2005; 65: 11459–11468., , et al.
- 21Bone marrow mesenchymal stem cells: historical overview and concepts. Hum Gene Ther 2010; 21: 1045–1056..
- 22Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2008; 2: 141–150., , et al.
- 23Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis. Cell Stem Cell 2012; 10: 544–555., , et al.
- 24Interplay between mesenchymal stem cells and lymphocytes: implications for immunotherapy and tissue regeneration. J Dent Res 2012; 91: 1003–1010., , .
- 25Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008; 371: 1579–1586., , et al.
- 26Ovariectomy disregulates osteoblast and osteoclast formation through the T-cell receptor CD40 ligand. Proc Natl Acad Sci U S A 2011; 108: 768–773., , et al.
- 27The role of recipient T cells in mesenchymal stem cell-based tissue regeneration. Int J Biochem Cell Biol 2012; 44: 2044–2050., , .
- 28Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-gamma and TNF-alpha. Nat Med 2011; 17: 1594–1601., , et al.
- 29Estrogen deficiency induces bone loss by enhancing T-cell production of TNF-alpha. J Clin Invest 2000; 106: 1229–1237., , et al.
- 30Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-gamma-induced class II transactivator. Proc Natl Acad Sci U S A 2003; 100: 10405–10410., , et al.
- 31Smad7 inhibits the survival nuclear factor kappaB and potentiates apoptosis in epithelial cells. Oncogene 2001; 20: 879–884., , et al.
- 32Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest 2001; 108: 807–816., , et al.
- 33A mechanism of suppression of TGF-beta/SMAD signaling by NF-kappa B/RelA. Genes Dev 2000; 14: 187–197., , et al.
- 34Interleukin-1beta up-regulation of Smad7 via NF-kappaB activation in human chondrocytes. Arthritis Rheum 2008; 58: 221–226., , et al.
- 35A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells. J Clin Invest 2004; 114: 1704–1713., , et al.
- 36Methylcholanthrene-induced sarcoma in singly fractured bone of mice. Tohoku J Exp Med 1967; 93: 125–129., , .
- 37Induction of murine fibrosarcomas by low dose treatment with 3-methylcholanthrene followed by promotion with 12-O-tetradecanoyl-phorbol-13-acetate. Cancer Lett 1984; 21: 317–324., , .
- 38Oct4 and Nanog directly regulate Dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells. Mol Cell 2012; 47: 169–82., , et al.
- 39Loss of metallothionein predisposes mice to diethylnitrosamine-induced hepatocarcinogenesis by activating NF-kappaB target genes. Cancer Res 2010; 70: 10265–10276., , et al.
- 40Multiple tumor types may originate from bone marrow-derived cells. Neoplasia 2006; 8: 716–724., , et al.
- 41Effect of aspirin and acetaminophen on proinflammatory cytokine-induced pain behavior in mice. Pharmacology 2005; 74: 152–156., , et al.
- 42Anti-inflammatory properties of pro-inflammatory interferon-gamma. Int Immunopharmacol 2003; 3: 1247–1255., .
- 43Identification of the homeobox protein Prx1 (MHox, Prrx-1) as a regulator of osterix expression and mediator of tumor necrosis factor alpha action in osteoblast differentiation. J Bone Miner Res 2011; 26: 209–219., , et al.
- 44Autocrine regulation of interferon gamma in mesenchymal stem cells plays a role in early osteoblastogenesis. Stem Cells 2009; 27: 550–558., , et al.
- 45Interferon gamma and T cells inhibit osteogenesis induced by allogeneic mesenchymal stromal cells. J Orthop Res 2013; 31: 227–234., , et al.
- 46TNF-alpha promotes fracture repair by augmenting the recruitment and differentiation of muscle-derived stromal cells. Proc Natl Acad Sci U S A 2011; 108: 1585–1590., , et al.
- 47Tumor necrosis factor inhibits mesenchymal stem cell differentiation into osteoblasts via the ubiquitin E3 ligase Wwp1. Stem Cells 2011; 29: 1601–1610., , et al.
- 48Estrogen deficiency increases the ability of stromal cells to support murine osteoclastogenesis via an interleukin-1and tumor necrosis factor-mediated stimulation of macrophage colony-stimulating factor production. J Biol Chem 1996; 271: 28890–28897., , et al.
- 49Synergy between interferon-gamma and tumor necrosis factor-alpha in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription 1 and nuclear factor kappaB. J Biol Chem 1997; 272: 14899–14907., , .
- 50TNFalpha and IFNgamma synergistically enhance transcriptional activation of CXCL10 in human airway smooth muscle cells via STAT-1, NF-kappaB, and the transcriptional coactivator CREB-binding. Protein J Biol Chem 2010; 285: 29101–29110., , et al.
- 51Synergistic activation of JNK/SAPK induced by TNF-alpha and IFN-gamma: apoptosis of pancreatic beta-cells via the p53 and ROS pathway. Cell Signal 2005; 17: 1516–1532., , et al.
- 52IFN-gamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death. J Immunol 2001; 166: 4481–4489., , et al.
- 53Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads. EMBO J 2001; 20: 4132–4142., , et al.
- 54NF-kappaB in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2002; 2: 301–310., , et al.
- 55TNF-alpha/NF-kappaB/Snail pathway in cancer cell migration and invasion. Br J Cancer 2010; 102: 639–644., .
- 56Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression. Nat Biotechnol 2002; 20: 587–591., , et al.
- 57Decreased IL-12 production and Th1 cell development by acetyl salicylic acid-mediated inhibition of NF-kappaB. Eur J Immunol 1998; 28: 3205–3213., , et al.
- 58Association between bone mineral density and the use of nonsteroidal anti-inflammatory drugs and aspirin: impact of cyclooxygenase selectivity. J Bone Miner Res 2003; 18: 1795–1802., , et al.
- 59Aspirin and NSAID use in older women: effect on bone mineral density and fracture risk. study of osteoporotic fractures research group. J Bone Miner Res 1996; 11: 29–35., , et al.
- 60Aspirin and other non-steroidal anti-inflammatory drugs and risk of non-hodgkin lymphoma. Cancer Epidemiol Biomarkers Prev. 2013. [Epub ahead of print], , et al.
- 61Cellular and molecular interactions of mesenchymal stem cells in innate immunity. Immunol Cell Biol 2013; 91: 27–31., .
Supporting Information
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Additional Supporting Information may be found in the online version of this article.
|Figure S1. Elevated levels of IFN-γ and TNF-α in OVX mice. Immunostaining showed increased IFN-γ and TNF-α positive cells (white arrows) in bone marrow and derma, but not in subcutaneous fat, in OVX mice when compared with sham-operated mice. Scale bars, 50 μm.
|Figure S2. The number of dermal MSCs is reduced in OVX mice. Immunohistochemical staining of MSC markers CD105, CD44 and CD73 showed markedly reduced number of dermal MSCs in OVX mice when compared with sham-operated mice. Scale bars, 50 μm.
|Figure S3. OVX results in trabecular bone deterioration and derma atrophy in mice. (A, B) H&E staining showed markedly thinner trabecular bone and reduced dermal thickness, respectively, in OVX mice when compared with sham-operated mice. n = 8 animals per group. *, P < 0.05. Scale bars, 50 μm.
|Figure S4. IFN-γ and TNF-α synergistically cause MSC deficiency in nude mice. (A, B) H&E staining showed that only IFN-γ/TNF-α treatment group showed marked loss of trabecular bone and derma atrophy. n = 8 animals per group. Scale bars, 100 μm.
|Figure S5. Local administration of IFN-γ/TNF-α causes MSC deficiency in nude mice. When IFN-γ and TNF-α, either alone or in combination, were subcutaneously transplanted with hydrogel as a controlled release system to nude mice, only IFN-γ–/TNF-α–infused group showed significantly reduced population doublings (A) and capacity to form mineralized nodules (B) in DMSCs. (C) H&E staining demonstrated that only IFN-γ/TNF-α locally transplanted with hydrogel showed significant derma atrophy. n = 5 animals per group. *, P < 0.05. Scale bars, 100 μm.
|Figure S6. IFN-γ and TNF-α impair MSC differentiation via NFκB/SMAD7 pathway. (A) Western blot showed upregulation of p-NFκBp65, p-IκB, SMAD7 and downregulation of p- SMAD1, RUNX2 and ALP in OVX-derived DMSCs. (B) TNF-α treatment showed a dosedependent NFκB activation. (C, D) Alizarin red staining showed that IFN-γ and TNF-α in combination but not alone caused a markedly reduction in mineralized nodule formation in DMSCs, with upregulated p-IκB, SMAD7 and down-regulated p-SMAD1, RUNX2 and ALP. (E, F) With knockdown of NFκB by IKKα siRNA, IFN-γ-/TNF-α-induced upregulation of SMAD7 and downregulation of p-SMAD1, RUNX2 and ALP were abolished, leading to the rescue of IFN-γ-/TNF-α–induced reduction of mineralized nodule formation in DMSCs. All experiments were performed in triplicate. *, P < 0.05.
|Figure S7. IFNGRα and TNFR1 are required for IFN-γ/TNF-α to snergistically induce activation of NFκB. (A) Combined knockdown of IFNGRα and TNFR1 resulted in markedly inhibition of IFN-γ–/TNF-α–induced activation of NFκBp65 and p-IκB when compared with TNFR1 knockdown group. (B) Knockdown of STAT-1 abolished TNF-α-/IFN-γ–induced activation of NFκB pathway. (C, D) Western blot analysis confirmed efficacy of siRNA in inhibiting IKKα, IFNGRα, TNFR1, STAT-1 and SMAD7.
|Figure S8. SMAD7 knockdown rescues impaired differentiation of BMMSCs and DMSCs. (A) Western blot showed that knockdown of SMAD7 downregulated p-SMAD1, RUNX2 and ALP in BMMSCs without downregulating p-IκB. (B) Alizarin red staining showed that knockdown of SMAD7 resulted in rescuing of IFN-γ-/TNF-α–induced reduction of mineralized nodule formation in BMMSCs. (C) Western blot showed that knockdown of SMAD7 downregulated RUNX2 and ALP in DMSCs without downregulating p-IκB. (D) Alizarin red staining showed that knockdown of SMAD7 resulted in rescuing of IFN-γ–/TNF-α–induced reduction of mineralized nodule formation in DMSCs. All experiments were performed in triplicate. *, P < 0.05.
|Figure S9. Long-term IFN-γ/TNF-α exposure increases susceptibility to MSC malignant transformation via NFκB–induced oncogenes. (A) Immunostaining of pan-CK showed negative cells in MCA–induced tumor in OVX mice. (B) Immunostaining of the tumors derived from s.c. inoculation of IFN-γ–/TNF-α–treated BMMSCs showed elevated expressions of VIM and PCNA, but negative expression of pan-CK. (C) Immunostaining of p-NFκB, c-FOS and c- MYC in bone marrow showed more positive cells in aging OVX mice. Scale bars, 50 μm.
|Figure S10. Aspirin rescues osteoporosis and derma atrophy in OVX mice. H&E staining showed that systemically applied aspirin rescued the deteriorated trabecular bone structures (A) and derma atrophy (B) in OVX mice. Scale bars, 100 μm. *, P < 0.05.
|Figure S11. Schematic diagram illustrating the mechanism in IFN-γ– and TNF-α–induced MSC deficiency and MSC tumorigenesis. Elevated levels of IFN-γ and TNF-α together, but not independently, synergistically cause MSC deficiency via NFκB–mediated activation of SMAD7, whereas long-term elevated levels of IFN-γ and TNF-α synergistically result in increased tendency toward MSC malignant transformation through NFκB–mediated upregulation of the oncogenes c-Fos and c-Myc.
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


text:
Top Definition
High quality crystal meth. used to discribe how clear or un-cut the meth is. Clear as glass shards.
Clinton's at it agian. He just went to the garage and snorted some shardicles.
de Slice Mcdell 20 Martie 2009
Trending Now
E-mailurile sunt trimise de la firstname.lastname@example.com. Nu-ți vom trimite mesaje nedorite.×

subset: commoncrawl


text:
Pizza Coupons
Select Local Merchants
Nexus CafeRead MoreNexus Cafe in Stanford serves traditional-meets-modern Asian-style cuisine from all over the continent. At this restaurant, kids of all ages are welcome. For comfortable outdoor service, Nexus Cafe sets up a seasonal patio. A relatively loud restaurant, this is not the place for a quiet night out. Ordering food? You can pick it up yourself! Drivers can find parking right by the restaurant, so don't forget your car keys. Nexus Cafe is what happens when cooks get creative with Asian cuisine, so get ready for pure deliciousness (and a medley of global flavors). Just know that when you're longing for a delicious hybrid of Asian cooking, Nexus Cafe is the ultimate casual dining spot.318 Campus DrStanford, CA US
Russo CafeRead MoreEnjoy a freshly tossed pizza loaded with toppings at Russo Cafe in Stanford. Bask in the sun (or moon!) light when you dine on Russo Cafe's outdoor patio. For no extra charge, utilize Russo Cafe's free wifi. For those in a rush, the pizzeria lets you take your food to go. At Russo Cafe, diners can score a guaranteed parking spot close to the restaurant. If you don't want a night that will cost you an arm and a leg but you do want a delicious meal, come to Russo Cafe. Select your toppings and create a delicious pizza made from scratch by visiting Russo Cafe.555 Salvatierra WalkStanford, CA US
New York PizzaRead MorePop over to New York Pizza for some hop (and highly-acclaimed) 'za, and find out what everyone's been raving about. Indulge in gluten-free and healthy menu options at New York Pizza. Ready for a drink to unwind? At this pizzeria, you can pair your meal with something from their full bar. The perfect place to take the kids, dining out at this pizzeria won't cost you a sitter. Groups of all sizes can easily be seated at New York Pizza. On warmer days, you can take advantage of New York Pizza's al fresco patio seating. You can't reserve a table at New York Pizza, so be sure to show up early. Jeans are just right for a meal at New York Pizza, which embraces a casual vibe. New York Pizza is known for serving great food, and they are able to serve it at your next event with their excellent catering. Turn your living room into a five-star restaurant with takeout or delivery from this pizzeria. At New York Pizza, you can count on quick and easy street parking close by. Bike parking is quick and easy at New York Pizza. Major credit cards — including Visa, MasterCard, Discover, and American Express — are accepted. Whether you're hungry first thing in the morning or prefer to eat a little later, New York Pizza is conveniently open for breakfast, lunch, and dinner. If pizza is your all-time favorite, it's important to find a pie that's worth your while. With star-studded reviews and sky-high ratings, there's no better way to spend your time than eating some 'za at New York Pizza. Don't feel like dressing up for dinner? No problem. New York Pizza's pizza is baked with top-notch ratings, so you can be sure to love your meal. New York Pizza cooks up great, casual pizzas just how you want them: delicious and scrumptious. Isn't it time you stopped trifling with average pizzas and went with the masters at New York Pizza?325 Hamilton AvenuePalo Alto, CA US
Pizz'a ChicagoRead MoreDeemed "pizza of the year" every year by Pizz'a Chicago's loyal fans, this deliciously-cheesy pizza will have you reaching for seconds, thirds, and even fourths. Cautious diners will appreciate the low-fat and gluten-free fare at Pizz'a Chicago. Order a bottle for the table if you like — this pizzeria has a full bar stocked with the best wine, beer, and more. Have a few picky young eaters in the family? Not a problem at this pizzeria, where the food and ambience are perfect for family dining. Reservations are not accepted at Pizz'a Chicago, so you may encounter a crowd during rush hours. Take it nice and easy at Pizz'a Chicago, where casual dress is the rule of the day. Love the food at this pizzeria but don't have the time to stay? You can pick up your food to eat when you're ready, or have them deliver straight to your home. Pizz'a Chicago will even bring the amazing food from their kitchen to yours. For no extra charge, diners can park in the connecting lot. Pizz'a Chicago makes bikers feel at ease with the multiple storage racks outside. Pizz'a Chicago's mid-priced fare will typically cost you about $30 per person or less. Easily charge your payment using one of many major credit card options. AM, midday, and PM meals are served at the pizzeria, but supper takes the cake for best in show. For talk-of-the-town pizza, Pizz'a Chicago is your pizzeria. Stop by for a slice or two and judge the ratings for yourself. So bring your appetite to Pizz'a Chicago. This no-muss, no-fuss pizza joint comes with rave reviews. Don't stress over planning a fancy dinner. Keep it fun and casual with a fresh, handmade pizza from Pizz'a Chicago. The pizza at Pizz'a Chicago is filled with endless flavors, so head on over today and enjoy a slice or two of yummy goodness.4115 El Camino RealPalo Alto, CA US
Howie's Artisan PizzaRead MoreWhether you prefer sausage, 'roni, or all-around veggie, Howie's Artisan Pizza's easy-to-please pizza has fans dishing out top-notch ratings. Drinks are also on the menu here, so diners can start the night off right. Little guys and gals will also love dining at this pizzeria, which offers a family-friendly environment (and menu). At Howie's Artisan Pizza, your large or small party can easily enjoy a meal. Dine under the sun (or stars) at Howie's Artisan Pizza with their charming outdoor seating. Lines can get long with no reservations, so be sure to plan for an early arrival. Howie's Artisan Pizza wants guests to dine in comfort, so save that stuffy suit for another date. The pizzeria also offers catering if you want to bring the flavors of Howie's Artisan Pizza to your next party or event. If you're in a hurry, place an order for pickup instead. We understand parking is expensive. That's why we've got a parking lot for you. Spend your money on our delicious food and drink. Make use of the safe and efficient bike parking at Howie's Artisan Pizza. Howie's Artisan Pizza is creating dishes any foodie will love at around $30. Roni, sausage, and veggie are just a few of the delicious options at Howie's Artisan Pizza. Taste the shining reviews for yourself when you head to Howie's Artisan Pizza for a tasty pizza pie. If you can't get enough pizza, be sure to try the pies at Howie's Artisan Pizza, which earn ratings too hot to handle. Don't stress over planning a fancy dinner. Keep it fun and casual with a fresh, handmade pizza from Howie's Artisan Pizza. When you need a good meal in a flash, grab a pizza from the highly-rated Howie's Artisan Pizza.855 El Camino RealPalo Alto, CA US
Patxi's Chicago PizzaRead MorePatxi's Chicago Pizza's piping pizza is just as hot as its ratings, and customers call this tasty spot one of the best around. Keep it low-fat and high-delicious at Patxi's Chicago Pizza. Beer, wine, and more are also available from this pizzeria's extensive drink list. Bring the whole family to this pizzeria, where kiddos are welcomed with open arms. Free wifi is available as well. Score quick and easy seating for your large group at Patxi's Chicago Pizza. You can't reserve a table ahead of time at Patxi's Chicago Pizza, so you may need to plan for a wait at prime times. Relaxed attire is perfectly fine at Patxi's Chicago Pizza, known for its laid-back ambience. The pizzeria also offers catering if you want to bring the flavors of Patxi's Chicago Pizza to your next party or event. If dining out is not on the agenda, this pizzeria offers delivery and pickup, too. Going out can be expensive. That's why we have our own free parking lot, so you spend your money on more fun things. Bike parking is quick and easy at Patxi's Chicago Pizza. Taste the greatness Patxi's Chicago Pizza is serving up with meals around $30. Breakfast, lunch, and dinner are all served at the pizzeria, but the dinner menu is the real standout. So come taste the pizza at Patxi's Chicago Pizza for yourself and see what all the ratings buzz is about. For a casual meal that is highly-rated, look no further than Patxi's Chicago Pizza's pizza. Patxi's Chicago Pizza cooks up great, casual pizzas just how you want them: delicious and scrumptious. When you are craving a little taste of Italy, make your way over to Patxi's Chicago Pizza and indulge in a fresh and flavorful pizza.441 Emerson StreetPalo Alto, CA US


text:
November 23, 2012
No answer yet
I travel in type systems.
Haskell is the most advanced purely-functional programming language. This group welcomes all functional programmers categorically! (or otherwise).
468 Hoodlums
3,762 Android Explorers
2,808 Clouds
837 Functionals
5,666 Developers & Entrepreneurs
10,706 Inner Circle
Meetup members, Log in
By clicking "Sign up" or "Sign up using Facebook", you confirm that you accept our Terms of Service & Privacy Policy


text:
|protein found in liver what abnormalities are these indicative of?
Jun 15, 2000
What test and what recommendations for high proteins counts and lypmolights....Pertaining to the liver and billirubin in normal and all others are in normal ranges?
Response from Dr. Dieterich
This is a little hard for me to understand. If the lymphocyte counts are high, there may a problem with your blood and you should see a hematologist. High proteins can also be a sign of disease and should be investigated. DTD
Milk Thistle effectiveness and Lamivudine as monotherapy
Is the HIV treatment necessary now?
- Flu Symptoms After Open Mouth Kissing Worried I Have HIV
- After Being Exposed To Hpv How Long Does It Take For Signs Of Cervical Cancer To Appear
- How Does Hydrogen Peroxide Cure Vaginal Infections?
- What Can Cause Vaginal Pain In A Toddler?
- Where Does Hpv Come From?
- Can You Get Hepatitis C From Vomit?
This forum is designed for educational purposes only, and experts are not rendering medical, mental health, legal or other professional advice or services. If you have or suspect you may have a medical, mental health, legal or other problem that requires advice, consult your own caregiver, attorney or other qualified professional.
Experts appearing on this page are independent and are solely responsible for editing and fact-checking their material. Neither TheBody.com nor any advertiser is the publisher or speaker of posted visitors' questions or the experts' material.
Review our complete terms of use and copyright notice.


text:
Are you having twins? Or have your delightful two arrived? How to cope with newborn twins can be a daunting thought. Here you can explore tips, facts and advice to get you through your pregnancy and that first all important year. From twin pregnancy to labor and delivery, newborn twins to breastfeeding twins and much more.
Twins Poll
Having twins is special and unique bringing big changes to your home and your life. Between sleepless nights, giggles and tickles with baby gear in every corner, routine and organization is the key for happy twins and happy parents.
Read and Share
Discovered you are expecting twins or have they arrived and are now on the road to raising twins? Here you can ask questions read stories or share your own twin experiences.
~ Moms Helping Moms ~
This site is just for you whether you're expecting two or you already have them, this site is great even for Grandparents, Aunts and family friends.
Search through this site to find information on all sorts of use information:
As a mother of identical twins I would like to share this journey with you as our beautiful baby twins grow and learn, enjoying every moment as we learn and grow right along with them.
Hi there, I'm Charlene firstly welcome to my web site.
My husband, John, and I are lucky enough to be raising twin girls and their wonderful older brother. Though it has been hard it gets easier as they get older, which comes with new and wonderful challenges.
Caring for twins is a tough job but so rewarding, amazing, joyful and all those good feelings...You can read more about us here
Jul 25, 16 06:09 PM
We just found out we are having twins, possibly 3!!! My son JUST turned 2 and we were already shocked at the thought of 1. I have so many thoughts, worries,
Jun 07, 16 12:34 AM
When I found out I was 5 1/2 weeks and I was scared only because this is my first pregnancy. But when I went into the doctors and I saw my babies I was
Apr 26, 16 06:18 PM
Cribs for twins, will it be one or two, mini and standard size or even a special crib made just for twins?
Like This Page?
Subscribe To Having Twins Today!
For Your Free Newsletter


text:
Welcome to WebmasterWorld Guest from 220.127.116.11
Forum Moderators: incrediBILL & martinibuster
joined:Feb 25, 2013
posts: 9
votes: 0
Google is GOING WAY OVERBOARD with their pounding and pounding on webmasters with their warnings, and scorecard BS. Do what we say! Do what we say! Do what we say!
joined:May 7, 2013
posts: 377
votes: 0


text:
Health Officials: America's Obesity Rates Soar
Health Officials: America's Obesity Rates Soar
133 results for 'survey'. Showing results 109 - 126
Health Officials: America's Obesity Rates Soar
CBN's 'Worldreach' Leading Nigerians to Christ
Survey: 1 in 9 Historic U.K. Churches at Risk
5.0-Magnitude Earthquake Shakes Canada
Strong Quake Rocks Eastern Indonesia
Philanthropy Declines 3.6 Percent in 2009
Gulf Oil Spill Slowing or Bigger than We Thought?
Survey: Euro's Future Looks Bleak
Survey: Americans Frugal on Valentine's Day
Poll Shows Americans Angry at Incumbents
Survey: Long Work Hours Takes Toll on Pastors
Survey: Christian Ministries Hit Hard by Recession
Powerful Quake Strikes Off Indonesian Island
Economists Predict Recovery to Remain Slow into '11
Earthquake Kills Hundreds in Western China
U.S. Troops Turning from Republican Party
Economists Believe Recession Will End in '09
Gallup Poll: Conservatism Dominant in U.S.


text:
Your cart is empty!
Sodium palmate, sodium palm kernelate, water, glycerin, melaleuca alternifolia (tea tree) leaf oil, melia azadirachta leaf extract, oat extract water, butylene glyco, avens ativa (oat) kernel extract, major leaf extract, sodium chloride, plantain extract, tetrasodium EDTA, iron oxides.
Use on affected areas to soothe itchy skin.
Reach Dentotape Unflavored Waxed Ribbon Dental Floss 100 Yards
Our Price:$2.63
Eucerin Calming Itch-Relief Treatment, Dry Skin Therapy - 6.8 Oz
Our Price:$6.78
Vanicream Cleansing Bar For Sensitive Skin, Fragrance-Free - 3.9 Oz
Our Price:$1.90
Sarna Anti-Itch Lotion For Sensitive Skin, Fragrance Free - 7.5 Oz
Our Price:$7.28
Sarna Original Anti-Itch Lotion - 7.5 Oz
Derma-E Itch Relief Lotion With Tea Tree, E And Chamomile, 6 Oz


text:
'The Clothesline Project' Promotes Domestic Violence Awareness
Written by Lindsey Watson
Last updated on October 22, 2013 @ 7:16PM
Created on October 22, 2013 @ 7:09PM
Every year groups all over the U.S. take part in a national art project, they do this by putting T-shirts on a clothesline as a memorial to the victims, and survivors of Domestic Violence.
Add your CommentYou must have an active WDTV.COM user account to post comments. Please login to your account, or create your free account today!
Comments (0)


text:
Copyright © Reed Business Information, a division of Reed Elsevier Inc. All rights reserved.
A Tear at the Edge of Creation: A Radical New Vision for Life in an Imperfect Universeby Marcelo Gleiser
View All Available Formats & Editions
- Overview
- Editorial Reviews
- Product Details
- Related Subjects
- Read an Excerpt
- What People Are Saying
- Meet the author
For millennia, shamans and philosophers, believers and nonbelievers, artists and scientists have tried to make sense of our existence by suggesting that everything is connected, that a mysterious Oneness binds us to everything else. People go to temples, churches, mosques, and synagogues to pray to their divine incarnation of Oneness. Following a surprisingly similar notion, scientists have long asserted that under Nature's apparent complexity there is a simpler underlying reality. In its modern incarnation, this Theory of Everything would unite the physical laws governing very large bodies (Einstein's theory of relativity) and those governing tiny ones (quantum mechanics) into a single framework. But despite the brave efforts of many powerful minds, the Theory of Everything remains elusive. It turns out that the universe is not elegant. It is gloriously messy.
Overturning more than twenty-five centuries of scientific thought, award-winning physicist Marcelo Gleiser argues that this quest for a Theory of Everything is fundamentally misguided, and he explains the volcanic implications this ideological shift has for humankind. All the evidence points to a scenario in which everything emerges from fundamental imperfections, primordial asymmetries in matter and time, cataclysmic accidents in Earth's early life, and duplication errors in the genetic code. Imbalance spurs creation. Without asymmetries and imperfections, the universe would be filled with nothing but smooth radiation.
A Tear at the Edge of Creation calls for nothing less than a new "humancentrism" to reflect our position in the universal order. All life, but intelligent life in particular, is a rare and precious accident. Our presence here has no meaning outside of itself, but it does have meaning. The unplanned complexity of humankind is all the more beautiful for its improbability. It's time for science to let go of the old aesthetic that labels perfection beautiful and holds that "beauty is truth." It's time to look at the evidence without centuries of monotheistic baggage. In this lucid, down-to-earth narrative, Gleiser walks us through the basic and cutting-edge science that fueled his own transformation from unifier to doubter—a fascinating scientific quest that led him to a new understanding of what it is to be human.
Editorial Reviews
Copyright © Reed Business Information, a division of Reed Elsevier Inc. All rights reserved.
"Peppered with personal anecdotes and wisdom from one of the science's most eloquent statesmen, this sweeping exploration of the imperfections at the heart of existence culminates in a hopeful message for humanity's self-fulfilling purpose in an otherwise meaningless universe."—Seed Magazine
Product Details
- 9781439127865
- Publisher:
- Free Press
- Publication date:
- 04/06/2010
- Sold by:
- Format:
- NOOK Book
- Pages:
- 304
- File size:
Read an Excerpt
A Tear at the Edge of CreationA Radical New Vision for Life in an Imperfect Universe
By Marcelo Gleiser
Free PressCopyright © 2010 Marcelo Gleiser
All right reserved.
There were no witnesses to what was about to happen. ?Happen? didn?t yet exist. Reality was timeless. Space also didn?t exist. The distance between two points was immeasurable. The points themselves could be anywhere, hovering and bouncing. Infinity tangled into itself. There was no here and now. Only Being.
Suddenly, a trembling, a vibration, an ordering began. Like roiling waves, space shuddered and swelled. What was near became far. What was now became past. As space and time were born, change began to happen: from Being to Becoming. Space bubbled; time unfurled. Soon, matter coalesced from the joint heaving of space and time, seeping out of its pores. This was no mundane substance: nothing like us; nothing like atoms.
This matter stretched space, made it inflate like a swelling balloon. This balloon became our Universe.
This is the creation story of our generation. The Holy Trinity here is Space, Time, and Matter. There is no Creator, no divine hand to guide the unfolding of the cosmos from Being to Becoming, from a timeless to an evolving state. The Universe happened on its own, a bubble of space that burst into existence from a sea of nothingness: creatio ex nihilo, creation out of nothing. That?s hard for us to fathom, as everything that we see happening seems to have a cause behind it. Should the Universe be any different? Could it really have emerged from nothing? Without a cause?
The first link in the long chain of causation from cosmic birth to now, the cause that started it all, is known traditionally as the First Cause. To do its job?trigger creation?it must necessarily be uncaused. The challenge, of course, is how to implement this mysterious, common-sense-violating, uncaused First Cause. Is science up to the task? Religions mostly use gods to bypass the creation dilemma. That works well for them, since physical laws and common sense don?t apply to gods. Being immortal, they are indifferent to causation: they exist, supernaturally, beyond time. In the book of Genesis, all-powerful, eternal God manipulated ?nothingness? with words, and there was light. For Jewish, Christian, and Muslim believers, He is the First Cause. All comes from Him, and He comes from nothing. It then follows that since He is perfect, what He creates must be perfect, too. Until, that is, Adam and Eve ate from the Tree of Knowledge and changed everything: curiosity and desire expelled us from Paradise, made us less than godly. Since then, as mere mortals, we?ve been aching to reconnect with what we?ve lost, to become one with God?s perfect creation. This noble-sounding quest has led us astray for too long. We need a fresh start.
According to some modern theories concerned with the origin of space, time, and matter, there exists a quantum nothingness, a bubbly foam of prototype universes called the ?multiverse? or the ?megaverse.? A few current theories state that the multiverse is eternal and hence uncaused. Occasionally, from the cosmic froth bubbles of space spring forth?baby universes. Some grow, while most shrink back to the nothingness whence they came. A clever interplay between gravity and matter allows baby universes to be born with zero energy cost: creation out of nothing. Time starts ticking when a bubble bursts into existence and begins to evolve, that is, when there is change to be accounted for. Multiverse theories propose that we live in one of these growing bubbles, one that emerged as randomly as a particle that is shot out of a radioactive atomic nucleus. Our bubble, our Universe with a capital U (to differentiate the portion of the universe we can actually measure from either hypothetical model universes or portions of the universe beyond our current measurements), has the apparently rare distinction of having existed long enough for galaxies, stars, and people to emerge: we result from the random birth of a highly improbable long-lived cosmos that has grown complex enough to spawn creatures capable of pondering their origins. This is a far cry from the premeditated supernatural creation portrayed in Genesis. But does it fully address the question of how everything came to be?
Clever as this scientific version of creation is in its attempt to do away with the First Cause, it still must be formulated according to accepted physical principles and laws: energy must be conserved; the speed of light and other fundamental constants of Nature must have the right values to ensure the viability of our Universe. Furthermore, a quantum nothingness, with its bubbly soup of prototype universes, is not quite what we would call the absence of everything. The point is, we humans cannot create something out of nothing. We need the materials; we need the joining rules. This limitation of ours is clearest when we try to make sense of the first of all creations, that of the Universe. Do not let claims to the contrary fool you, even if they involve awe-inspiring terms like quantum vacuum decay, string landscape, extra-dimensional space-time, or multi-brane collision: we are far from having a convincing, empirically validated (meaning tested or even testable) scientific narrative of creation. Even if, one day, we do devise such a theory, it must always be qualified as a scientific theory of creation, based on a series of assumptions.
Science needs a framework, a scaffolding of principles and laws, to operate. It cannot explain everything, because it needs to start with something. This something must be taken for granted. Examples of such starting points are the axioms of mathematical theorems?unproved statements accepted as being self-evident and thus supposedly true?and, in physical theories, a number of laws of Nature, such as energy and electric charge conservation, that often are extrapolated to be valid well beyond their tested range. Seeing how well these laws work for the natural phenomena we can observe and measure, we assume that they held in the extreme environment prevalent near the Big Bang, the event that marked the beginning of time. But we cannot be sure?and scientists should never claim they are?until there is clear experimental confirmation. ?Extraordinary claims need extraordinary evidence,? noted University of California paleontologist J. William Schopf.
On the other hand, modern cosmological theories do explain the physical processes that took place very close to the beginning of time, an achievement that is?and should be loudly advertised as?truly amazing. We can now state with confidence that the Universe did spring from a hot and dense soup of elementary particles a little under 14 billion years ago, even though we still don?t know how the spring sprang. We know that the young, minute-old cosmos forged the lightest chemical elements, and that exploding stars forged?and continue to forge?the heavier ones needed for life. We understand the workings of the genetic code and the mechanism behind the staggering variety of animals and plants on Earth. Barring the existence of other self-aware beings capable of theorizing about life and death, we?imperfect accidents of creation?are how the Universe thinks about itself. To my mind, this is a life-transforming revelation, the substance of this book. Even though we live in no special place in the cosmos and play no starring role in the grand scheme of things, the fact that we carry this banner?alone or not?does make us very special. For this very reason, we must be extra careful. In spite of all our achievements, we will do well to remember that our story is just our story, imperfect and limited as we are; we will do well to remember not to go after absolute truth but after understanding. As Tom Stoppard reminds us in his play Arcadia, it?s not knowing everything, but wanting to know that matters.
Wonderful as it is, science is a human construction, a narrative we create to make sense of the world around us. The ?truths? that we obtain, such as Newton?s universal law of gravitation or Einstein?s special theory of relativity, are indeed impressive, but always of limited validity. There is always more to explain beyond the reach of a theory. New scientific revolutions are going to happen. World-views will shift. Yet, vain as we are, we place too much weight on our achievements. Our successes have led us to believe that these partial truths are scattered pieces of a single puzzle, the components of a Final Truth, waiting to be discovered. Great minds of the distant and recent past have devoted decades of their lives in search of this Holy Grail, Nature?s hidden code: Pythagoras, Aristotle, Kepler, Einstein, Planck, Schr?dinger, Heisenberg. The list is long. Thousands more are doing so today. Knowingly or unknowingly, they are heirs to a philosophical tradition rooted in ancient Greece that links perfection and beauty with truth. Over the centuries, this tradition was fused with monotheistic belief: God?s creation was perfect and beautiful. To understand it, to search for immortal truth, became the highest of aspirations. Since the birth of modern science in the early 1600s, a passion akin to religious fervor has led to the widespread conviction that the puzzle can be solved, that we are closer than ever, that Nature?s hidden code will soon be unveiled in all its glory. British physicist Stephen Hawking, as many before him, metaphorically compared the achievement to ?knowing the mind of God.? But is that so? Are we truly getting any closer? Or are we lost, searching for the unattainable? Should we instead be asking why we need to believe so badly in this Final Truth? Should we be asking why we are so convinced that it is there to be discovered? Does the experimental and observational evidence at hand truly point this way? Or is this Final Truth simply the scientific incarnation of the monotheistic tradition of the West, a yearning for a God that reason exorcised from spiritual life?
Given that the Final Truth necessarily explains the origin of the Universe, we now see how these two quests are one and the same: the Final Truth contains the First Cause; the First Cause contains the Final Truth. Can we, limited beings that we are, explain creation in all of its astonishing complexity?
We know at least two answers:
?Sure!? exclaim the Unifiers. ?There is a fundamental set of physical laws, writ deep into Nature?s essence, behind all there is. Given time, we will uncover these laws and make sense of it all. Together, these laws are the embodiment of the unified field theory, the supreme expression of the hidden mathematical symmetry of Nature. We call it the Theory of Everything.?
?Sure!? exclaim the Believers. ?We already know all the answers. They are written in our Holy Book. Creation is the work of our all-powerful God. Only a supernatural power could exist before space. Only a supernatural power could be before time. Only a supernatural power could transcend material reality to create it.?
Are we limited to these two choices? Is there a third alternative? For millennia, we have lived under the mythic spell of the One. Kneeling at our temples or searching for the mathematical ?mind of God,? we have yearned for a connection with what is beyond the merely human; we have dreamt of an abstract perfection that we could not find in our lives. In doing so, we closed our eyes to ourselves, refusing to accept the fragility of our existence. It is now time to move on. It is now time to shake free of the old imperative for perfection and embrace the lessons of a new scientific worldview that explores the creative power of Nature?s imperfections and accepts that there are limits to knowledge.
The journey will be humbling, as we face the smallness of our existence in a vast, indifferent cosmos. And yet, small that we are, our very existence makes us unique. Thinking aggregates of inanimate atoms, we are rare and precious. In a few millennia, we have achieved the power to change the course of our planet?s history and, with it, our own. Humanity is at a crossroads. The decisions we make now will shape our future and that of our planet. It is time to understand that preserving life is what really matters.
? 2010 Marcelo Gleiser
Excerpted from A Tear at the Edge of Creation by Marcelo Gleiser Copyright © 2010 by Marcelo Gleiser. Excerpted by permission.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.
What People are saying about this
Meet the Author
MARCELO GLEISER is a professor of physics and astronomy at Dartmouth College where he runs a very active cosmology group. His work in theoretical physics focuses on the dynamical processes that took place during the very early universe. As such, it brings together particle physics, the study of the very small, and cosmology, the study of the very large. He is the author of The Dancing Universe: From Creation Myths to the Big Bang; and The Prophet and the Astronomer.
Customer Reviews
Average Review:
and post it to your social network
Most Helpful Customer Reviews
See all customer reviews >


text:
Upload Photo
Countdown: Top 10 Islanders of all-time
Newsday's Arthur Staple counts down the 10 best players to put on the skates for the New York Islanders.
Newsday.com now uses Facebook for our comment boards. Please read our guidelines and connect your Facebook account to comment.
Yankees vs. Astros 25 Olympians to follow on Twitter ahead of Rio In the face! The 3,000 hits club 2016 PGA Championship practice rounds Eagles training camp photos Fight photos: Terence Crawford vs. Viktor Postol This day in sports history: July 25 New York State Open USA vs. China Brickyard 400 Top 20 fantasy football running backs


text: I've started working also on UML/2.4. Today I've split the changes between 2.4.24-1um and 2.4.24-2um into several patches; the HUMFS ones are carefully split out, but I've separated even the others. Now I need testers. Lots of people cannot use UML patches later than 2.4.24-1um, since they contain a lot of Hostfs bugs and some stability issues. Now, if you use just some patches, you'll avoid the bugs in the other. You should apply surely the first 3 patches, IMHO; the other ones should not hurt; inside txt/ there is a description of them. mini-changes.patch Various little changes which don't hurt. use_setjmp_wrapper.patch Adds setjmp_wrapper and use it, to fix klogd bug. use-gettimeofday.patch Replace rdtsc with gettimeofday. remapper-fix.patch Fixes probably to the ubd-mmap code SIG-fileHandle-changes.patch Cope with signals from various places. exec-shield.patch Patch for exec-shield users (IMHO unsafe) expand-os-for-HUMFS.patch Expands os_ layer, seems to be for HUMFS only. HUMFS.patch Adds HUMFS support, but create problems for Hostfs The actual patches can be found inside the patches/ subdir in the tarball, and the "series" file contains the order. THEY ARE AGAINST 2.4.24-1uml. If you want to apply it onto 2.4.26, don't forget to include the following patch (just for 2.4.26): http://vs165141.v.sectoor.de/uml-2.4.26-cmpxchg.patch But you should move the content of 2.4.24-2-inc-split/ folder into the root of the linux tree (i.e. under Uml-linux) and then run "pushpatch 30" after installing patch-scripts. Here there is the tarball: http://www.user-mode-linux.org/~blaisorblade/patches/2.4.24-2-inc-split.tar.bz2 Here you can download patch-scripts; to install them you must just expand the tarball into /usr/local/bin (or any folder you like; remember to update the path if needed). http://www.zip.com.au/~akpm/linux/patches/patch-scripts-0.18/ Bye -- Paolo Giarrusso, aka Blaisorblade Linux registered user n. 292729


text:
Making Music: David Longstreth
According to the indie pop-orchestral-synth band Dirty Projectors' MySpace page, Dave Longstreth doesn't play an instrument or write the songs; he's the group's "musical director." That catchall title would probably be best interpreted to mean that he's the guy responsible for the band's lush, eerie, pop sounds that somehow have listeners tapping their toes while feeling haunted. Longstreth will be in the hot seat at the Walker's Maguire Theater, taking questions from James Everest of Vicious Vicious and Roma di Luna. Longstreth will also show off his skills through musical demonstrations and videos, and fans in the audience will have a chance to pick his brain. Stick around after the interview for a set by Radio K's DJ Paul Harding and a reception in the museum's Art Lab.
Thu., March 5, 7 p.m., 2009
Get the Things to Do Newsletter
Sign up for our weekly guide to events in Minneapolis & St. Paul, and never be bored again. With suggestions for every day of the week, our recommendations will keep you busy on any budget.
Popular Stories
Sponsor Content


text:
I'm using this plugin (which is awesome, btw!), including a custom template, to display a custom post type of special offers in my site's sidebar. The CPT includes a metabox for the offer's status (i.e., Active/Inactive).
I'm wondering if there's any code I can add to the custom template to further filter the widget's query by the status? I've been able to do this successfully in other areas of the site (like so: http://pastebin.com/nGzdrLUK), but I'm unsure if or how to filter a query that's already been called. I tried using pre_get_posts but couldn't make it work.


text:
The Mt Sinai School of Medicine of the City University of New York has a retention program for disadvantaged medical students. The program has been functional for two years and was instituted because of multiple course failures among its disadvantaged students. The program consists of a summer enrichment unit and formal tutorials during the academic year. Our two-year experience demonstrates a complete reversal in the academic performance of these students.
Some tools below are only available to our subscribers or users with an online account.
Download citation file:
Customize your page view by dragging & repositioning the boxes below.
More Listings atJAMACareerCenter.com >
Enter your username and email address. We'll send you a link to reset your password.
Enter your username and email address. We'll send instructions on how to reset your password to the email address we have on record.
Need assistance?
Athens and Shibboleth are access management services that provide single sign-on to protected resources. They replace the multiple user names and passwords necessary to access subscription-based content with a single user name and password that can be entered once per session. It operates independently of a user's location or IP address. If your institution uses Athens or Shibboleth authentication, please contact your site administrator to receive your user name and password.


text:
good animal feed fish meal
1 Twenty-Foot Container (Min. Order)
Good quality cooking Peanut Oil
US $450-1000 / Ton
25 Tons (Min. Order)
White Maize/ Corn For Sale Good Price
US $0-1 / Metric Ton
1 Forty-Foot Container (Min. Order)
South African good quality peanut in shell ,the biggest groundnut factory
US $500-1200 / Metric Ton
18 Tons (Min. Order)
High quality 99.5% Diisopropylamine with good price
US $2000.0-2000.0 / Metric Tons
20 Metric Tons (Min. Order)
Good price Methylene chloride for sale
US $500.0-500.0 / Metric Tons
400 Metric Tons (Min. Order)
Good Quality TAPIOCA CHIP/CASSAVA CHIP for Animal feed
US $100-180 / Ton
1 Twenty-Foot Container (Min. Order)
Good taste natural chesnut for sale
US $550-800 / Ton
15 Metric Tons (Min. Order)
Go to Page
Prev 1 Next
Haven't found the right supplier yet ? Let matching verified suppliers find you. Get Quotation NowFREE
Do you want to show imported goods or other products of your own company? Display your Products FREE now!
Product Features
Supplier Features
Supplier Types
Recommendation for you
related suppliers