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Scientific Reports Jul 2020Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through...
Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through direct actions on cells of the myeloid lineage. Here, we investigated the molecular mechanism of this effect. We report that 17β-estradiol (E) decreased osteoclast number by promoting the apoptosis of early osteoclast progenitors, but not mature osteoclasts. This effect was abrogated in cells lacking Bak/Bax-two pro-apoptotic members of the Bcl-2 family of proteins required for mitochondrial apoptotic death. FasL has been previously implicated in the pro-apoptotic actions of E. However, we show herein that FasL-deficient mice lose bone mass following ovariectomy indistinguishably from FasL-intact controls, indicating that FasL is not a major contributor to the anti-osteoclastogenic actions of estrogens. Instead, using microarray analysis we have elucidated that ERα-mediated estrogen signaling in osteoclast progenitors decreases "oxidative phosphorylation" and the expression of mitochondria complex I genes. Additionally, E decreased the activity of complex I and oxygen consumption rate. Similar to E, the complex I inhibitor Rotenone decreased osteoclastogenesis by promoting osteoclast progenitor apoptosis via Bak/Bax. These findings demonstrate that estrogens decrease osteoclast number by attenuating respiration, and thereby, promoting mitochondrial apoptotic death of early osteoclast progenitors.
Topics: Adenosine Triphosphate; Animals; Apoptosis; Biomarkers; Bone Density; Bone and Bones; Cell Count; Cell Differentiation; Cells, Cultured; Estrogens; Female; Gene Expression Regulation; Mice; Mice, Knockout; Mitochondria; Monocyte-Macrophage Precursor Cells; Osteoclasts; Osteogenesis; Oxidative Phosphorylation; Signal Transduction
PubMed: 32686739
DOI: 10.1038/s41598-020-68890-7 -
Pharmacological Research Jul 2019Poly(ADP-ribose) polymerase (PARP) is involved in the pathogenesis of cell dysfunction, inflammation and organ failure during septic shock. The goal of the current study...
The PARP inhibitor olaparib exerts beneficial effects in mice subjected to cecal ligature and puncture and in cells subjected to oxidative stress without impairing DNA integrity: A potential opportunity for repurposing a clinically used oncological drug for the experimental therapy of sepsis.
Poly(ADP-ribose) polymerase (PARP) is involved in the pathogenesis of cell dysfunction, inflammation and organ failure during septic shock. The goal of the current study was to investigate the efficacy and safety of the clinically approved PARP inhibitor olaparib in experimental models of oxidative stress in vitro and in sepsis in vivo. In mice subjected to cecal ligation and puncture (CLP) organ injury markers, circulating and splenic immune cell distributions, circulating mediators, DNA integrity and survival was measured. In U937 cells subjected to oxidative stress, cellular bioenergetics, viability and DNA integrity were measured. Olaparib was used to inhibit PARP. The results show that in adult male mice subjected to CLP, olaparib (1-10 mg/kg i.p.) improved multiorgan dysfunction. Olaparib treatment reduced the degree of bacterial CFUs. Olaparib attenuated the increases in the levels of several circulating mediators in the plasma. In the spleen, the number of CD4+ and CD8+ lymphocytes were reduced in response to CLP; this reduction was inhibited by olaparib treatment. Treg but not Th17 lymphocytes increased in response to CLP; these cell populations were reduced in sepsis when the animals received olaparib. The Th17/Treg ratio was lower in CLP-olaparib group than in the CLP control group. Analysis of miRNA expression identified a multitude of changes in spleen and circulating white blood cell miRNA levels after CLP; olaparib treatment selectively modulated these responses. Olaparib extended the survival rate of mice subjected to CLP. In contrast to males, in female mice olaparib did not have significant protective effects in CLP. In aged mice olaparib exerted beneficial effects that were less pronounced than the effects obtained in young adult males. In in vitro experiments in U937 cells subjected to oxidative stress, olaparib (1-100 μM) inhibited PARP activity, protected against the loss of cell viability, preserved NAD levels and improved cellular bioenergetics. In none of the in vivo or in vitro experiments did we observe any adverse effects of olaparib on nuclear or mitochondrial DNA integrity. In conclusion, olaparib improves organ function and extends survival in septic shock. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of septic shock.
Topics: Animals; Anti-Inflammatory Agents; Cecum; Cytokines; DNA; Drug Repositioning; Female; Humans; Ligation; Liver; Lung; Lymphocyte Count; Male; Mice, Inbred C57BL; Oxidative Stress; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Punctures; Sepsis; Spleen; U937 Cells
PubMed: 31071432
DOI: 10.1016/j.phrs.2019.104263 -
Journal of Experimental & Clinical... Nov 2012Epidemiological studies revealed significantly lower mortality rates in cancer patients receiving cardiac glycosides, which turned on interest in the anticancer...
BACKGROUND
Epidemiological studies revealed significantly lower mortality rates in cancer patients receiving cardiac glycosides, which turned on interest in the anticancer properties of these drugs. However, cardiac glycosides have also been shown to stimulate cell growth in several cell types. In the present investigation we analyzed the pro-death and pro-survival properties of ouabain in the human lymphoma derived cell line U937.
METHODS
ROS, intracellular Ca++, cell cycle were evaluated by loading the cells with fluorescent probes under cytofluorimetry. Cell counts and evaluation of trypan blue-excluding cells were performed under optic microscope. Protein detection was done by specific antibodies after protein separation from cellular lysates by SDS-PAGE and transfer blot.
RESULTS
High doses of ouabain cause ROS generation, elevation of [Ca++]i and death of lymphoma derived U937 cells. Lower doses of OUA activate a survival pathway in which plays a role the Na+/Ca++-exchanger (NCX), active in the Ca++ influx mode rather than in the Ca++ efflux mode. Also p38 MAPK plays a pro-survival role. However, the activation of this MAPK does not appear to depend on NCX.
CONCLUSION
This investigation shows that the cardiac glycoside OUA is cytotoxic also for the lymphoma derived cell line U937 and that can activate a survival pathway in which are involved NCX and p38 MAPK. These molecules can represent potential targets of combined therapy.
Topics: Calcium; Cell Death; Cell Survival; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Lymphoma; Ouabain; Reactive Oxygen Species; Sodium-Calcium Exchanger; U937 Cells; p38 Mitogen-Activated Protein Kinases
PubMed: 23153195
DOI: 10.1186/1756-9966-31-95 -
The Journal of Allergy and Clinical... May 2014Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in... (Clinical Trial)
Clinical Trial
The eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1): a novel therapeutic target for eosinophilic disorders.
BACKGROUND
Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in all subjects. Additional approaches are clearly needed.
OBJECTIVE
We sought to explore the potential of the human eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1) as a therapeutic target for eosinophilic disorders.
METHODS
EMR1 expression was assessed in blood and bone marrow specimens from eosinophilic and healthy subjects, cell lines, CD34(+) cells differentiated in vitro, and tissue biopsy specimens by using flow cytometry, quantitative PCR, and immunostaining. Eosinophil targeting by a novel, humanized, afucosylated anti-EMR1 IgG1 was evaluated in vitro by using a natural killer cell-mediated killing assay and in vivo in cynomolgus monkeys.
RESULTS
Analysis of blood and bone marrow cells from healthy and eosinophilic donors and in vitro-differentiated CD34(+) cells confirmed restriction of human EMR1 surface and mRNA expression to mature eosinophils. Tissue eosinophils also expressed EMR1. Although EMR1 was highly expressed on eosinophils from all subjects, surface expression was negatively correlated with absolute eosinophil counts (r = -0.46, P < .001), and soluble plasma levels correlated positively with absolute eosinophil counts (r = 0.69, P < .001), suggesting modulation of EMR1 in vivo. Nevertheless, afucosylated anti-EMR1 mAb dramatically enhanced natural killer cell-mediated killing of eosinophils from healthy and eosinophilic donors and induced a rapid and sustained depletion of eosinophils in monkeys.
CONCLUSION
EMR1 expression is restricted to mature blood and tissue eosinophils. Targeting of eosinophils with afucosylated anti-EMR1 antibody shows promise as a treatment for eosinophilic disorders.
Topics: Antibodies, Monoclonal, Murine-Derived; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Calcium-Binding Proteins; Eosinophilia; Eosinophils; Female; Gene Expression Regulation; Humans; Immunoglobulin G; K562 Cells; Male; Membrane Glycoproteins; Mucins; Receptors, G-Protein-Coupled; U937 Cells
PubMed: 24530099
DOI: 10.1016/j.jaci.2013.11.041 -
Contrast Media & Molecular Imaging Nov 2016Cellular MRI, which visualizes magnetically labelled cells (cells*), is an active research field for in vivo cell therapy and tracking. The simultaneous relaxation rate...
Cellular MRI, which visualizes magnetically labelled cells (cells*), is an active research field for in vivo cell therapy and tracking. The simultaneous relaxation rate measurements (R *, R , R ) are the basis of a quantitative cellular MRI method proposed here. U937 cells were labelled with Molday ION Rhodamine B, a bi-functional superparamagnetic and fluorescent nanoparticle (U937*). U937* viability and proliferation were not affected in vitro. In vitro relaxometry was performed in a cell concentration range of [2.5 × 10 -10 ] cells/mL. These measurements show the existence of complementary cell concentration intervals where these rates vary linearly. The juxtaposition of these intervals delineates a wide cell concentration range over which one of the relaxation rates in a voxel of an in vivo image can be converted into an absolute cell concentration. The linear regime was found at high concentrations for R in the range of [10 - 2 × 10 ] cells/mL, at intermediate concentrations for R in [2.5 × 10 - 5 × 10 ] cells/mL and at low concentrations for R * in [8 × 10 - 5 × 10 ] cells/mL. In vivo relaxometry was performed in a longitudinal study, with labelled U937 cells injected into a U87 glioma mouse model. Using in vitro data, maps of in vivo U937* concentrations were obtained by converting one of the in vivo relaxation rates to cell concentration maps. MRI results were compared with the corresponding optical images of the same brains, showing the usefulness of our method to accurately follow therapeutic cell biodistribution in a longitudinal study. Results also demonstrate that the method quantifies a large range of magnetically labelled cells*. Copyright © 2016 John Wiley & Sons, Ltd.
Topics: Animals; Brain; Cell Count; Cell Movement; Cell Transplantation; Fluorescence; Glioma; Heterografts; Humans; Magnetic Resonance Imaging; Magnetics; Mice; U937 Cells
PubMed: 27766757
DOI: 10.1002/cmmi.1715 -
Virology May 1999The ability of HIV to match levels of viral mRNA to the activation state of the host cell may play a role in its ability to persist as well as to replicate. This linkage...
The ability of HIV to match levels of viral mRNA to the activation state of the host cell may play a role in its ability to persist as well as to replicate. This linkage depends on the function of the viral transcriptional regulatory protein, Tat, which increases the efficiency of RNA elongation (transcriptional processivity) in response to cellular activation. To quantify levels of Tat function in vivo, a quantitative competitive RT-PCR assay was developed that reflects levels of TAR leader fragments (nonprocessive transcripts) and viral mRNA (processive transcripts), indicating low or high levels of Tat function, respectively. The abundance of these RNA species was measured in peripheral blood mononuclear cells (PBMC) of 22 HIV-1-positive individuals (CD4(+) T cell counts 63-934/mm3) and in established cell line models of HIV constitutive replication (H9IIIB) and reversible latency (U1 and ACH-2). In PBMC, the level of total viral transcripts ranged over four orders of magnitude; however, nonprocessive transcription predominated: 70% of PBMC samples had a ratio of processive to total transcripts of <0.3 and none of the samples had 100% processivity. The cell line studies revealed that, even in activated H9IIIB cells, nonprocessive transcription dominates and that latently infected cells can have different transcriptional responses to activation. This is the first study that enumerates degrees of transcriptional processivity in the circulating mononuclear cell compartment and the results suggest that limitation of Tat function may be a common phenotype throughout the course of the disease.
Topics: Cell Line; Gene Expression Regulation, Viral; Gene Products, tat; HIV-1; Humans; Leukocytes, Mononuclear; Mitogens; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Viral; T-Lymphocytes; Transcription, Genetic; U937 Cells; tat Gene Products, Human Immunodeficiency Virus
PubMed: 10329550
DOI: 10.1006/viro.1999.9647 -
Medical Science Monitor : International... Oct 2019BACKGROUND Acute myeloid leukemia (AML) is associated with a high relapse rate and poor prognosis. This study aimed to use weighted gene coexpression network analysis...
Weighted Gene Coexpression Network Analysis Identifies Cysteine-Rich Intestinal Protein 1 (CRIP1) as a Prognostic Gene Associated with Relapse in Patients with Acute Myeloid Leukemia.
BACKGROUND Acute myeloid leukemia (AML) is associated with a high relapse rate and poor prognosis. This study aimed to use weighted gene coexpression network analysis (WGCNA) of gene coexpression networks to identify candidate prognostic biomarker genes in patients with AML and to investigate the expression of these genes in the human U937 cell line in vitro. MATERIAL AND METHODS RNA-seq data were retrieved from the Cancer Genome Atlas (TCGA) and included bone marrow samples and survival data of patients with AML (N=151), patients who did not relapse after treatment (N=119), and patients with relapse (N=40). Differentially expressed genes were identified, WGCNA was used to detect functional modules, and survival analysis was performed. The Cell Counting Kit-8 (CCK-8) assay investigated the proliferation of U937 cells transfected with short hairpin RNAs (shRNAs), shCRIP1, shHIST1H1C, and shHIST1H1E. RNA-seq analysis identified gene expression following CRIP1 knockdown. RESULTS Eighty-two genes were associated with both relapse and prognosis in patients with AML. There were two prognosis-related gene modules in the coexpression network. In the coexpression network, the histone cluster 1 H1 family member gene, HIST1H1C had the maximum relapse fold change, HIST1H1E had the lowest survival p-value, and the cysteine-rich intestinal protein 1 (CRIP1) gene had the most edge numbers and was significantly associated with poor prognosis (P=0.0165786). RNA-seq data showed that there was a significant difference in gene expression after CRIP1 knockdown in U937 cells. CONCLUSIONS WGCNA of gene coexpression networks identified CRIP1 as a potential prognostic biomarker gene in patients with AML.
Topics: Carrier Proteins; Cell Proliferation; China; Computational Biology; Databases, Genetic; Female; Gene Expression Profiling; Gene Regulatory Networks; Humans; LIM Domain Proteins; Leukemia, Myeloid, Acute; Male; Prognosis; RNA, Long Noncoding; RNA, Small Interfering; Recurrence; U937 Cells
PubMed: 31577790
DOI: 10.12659/MSM.918092 -
Journal of Toxicology and Environmental... 2013This study detailed the sequence of recurring inflammatory events associated with episodic allergen exposures of mice resulting in airway hyperreactivity, sustained...
This study detailed the sequence of recurring inflammatory events associated with episodic allergen exposures of mice resulting in airway hyperreactivity, sustained inflammation, goblet-cell hyperplasia, and fibrogenesis that characterize a lung with chronic asthma. Ovalbumin (OVA)-sensitized female BALB/c mice were exposed to saline-control or OVA aerosols for 1 h per day for episodes of 3 d/wk for up to 8 wk. Lung inflammation was assessed by inflammatory cell recoveries using bronchoalveolar lavages (BAL) and tissue collagenase dispersions. Cell accumulations were observed within airway submucosal and associated perivascular spaces using immunohistochemical and tinctorial staining methods. Airway responsiveness to methacholine aerosols were elevated after 2 wk and further enhanced to a sustained level after wk 4 and 8. Although by wk 8 diminished OVA-induced accumulations of eosinophils, neutrophils, and monocyte-macrophages were observed, suggesting diminished responsiveness, the BAL recovery of lymphocytes remained elevated. Airway but not perivascular lesions persisted with a proliferating cell population, epithelial goblet-cell hyperplasia, and evidence of enhanced collagen deposition. Examination of lung inflammatory cell content before the onset of the first, second, and fourth OVA exposure episodes demonstrated enhancements in residual BAL lymphocyte and BAL and tissue eosinophil recoveries with each exposure episode. Although tissue monocyte-macrophage numbers returned to baseline prior to each exposure episode, the greatest level of accumulation was observed after wk 4. These results provide the basis for establishing the inflammatory and exposure criteria by which episodic environmental exposures to allergen might result in the development of a remodeled lung in asthma.
Topics: Aerosols; Allergens; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cell Count; Chronic Disease; Collagen; Female; Fibrosis; Inhalation Exposure; Leukocytes; Lung; Methacholine Chloride; Mice; Mice, Inbred BALB C; Monocyte-Macrophage Precursor Cells; Ovalbumin; Recurrence; Respiratory Function Tests; Time Factors
PubMed: 23356647
DOI: 10.1080/15287394.2013.752323 -
Ultrasonics Sonochemistry Jul 2016In this study, we report on the potential use of platinum nanoparticles (Pt-NPs), a superoxide dismutase (SOD)/catalase mimetic antioxidant, in combination with 1MHz...
In this study, we report on the potential use of platinum nanoparticles (Pt-NPs), a superoxide dismutase (SOD)/catalase mimetic antioxidant, in combination with 1MHz ultrasound (US) at an intensity of 0.4 W/cm(2), 10% duty factor, 100 Hz PRF, for 2 min. Apoptosis induction was assessed by DNA fragmentation assay, cell cycle analysis and Annexin V-FITC/PI staining. Cell killing was confirmed by cell counting and microscopic examination. The mitochondrial and Ca(2+)-dependent pathways were investigated. Caspase-8 expression and autophagy-related proteins were detected by spectrophotometry and western blot analysis, respectively. Intracellular reactive oxygen species (ROS) elevation was detected by flow cytometry, while extracellular free radical formation was assessed by electron paramagnetic resonance spin trapping spectrometry. The results showed that Pt-NPs exerted differential effects depending on their internalization. Pt-NPs functioned as potent free radical scavengers when added immediately before sonication while pre-treatment with Pt-NPs suppressed the induction of apoptosis as well as autophagy (AP), and resulted in enhanced cell killing. Dead cells displayed the features of pyknosis. The exact mode of cell death is still unclear. In conclusion, the results indicate that US-induced AP may contribute to cell survival post sonication. To our knowledge this is the first study to discuss autophagy as a pro-survival pathway in the context of US. The combination of Pt-NPs and US might be effective in cancer eradication.
Topics: Apoptosis; Humans; Lymphoma; Metal Nanoparticles; Platinum; U937 Cells
PubMed: 26964942
DOI: 10.1016/j.ultsonch.2015.12.013 -
The Journal of Experimental Medicine Oct 1970The mononuclear phagocytes of the bone marrow can be classified into two cell types, promonocytes and monocytes. The present study was performed to establish whether the...
The mononuclear phagocytes of the bone marrow can be classified into two cell types, promonocytes and monocytes. The present study was performed to establish whether the promonocytes are the progenitors of the monocytes and to determine the kinetic characteristics of the promonocytes and monocytes in the bone marrow compartment. Both in vitro labeling studies with thymidine-(3)H and determination of the relative amount of DNA in the nuclei of individual cells showed that under normal steady-state conditions the promonocytes are proliferating cells and the monocytes, nondividing cells. In vivo labeling studies provided further evidence that the promonocytes are the progenitor cells of the monocytes. During the first 24 hr after labeling, the promonocytes showed a constant high level of labeling (about 70%). The mean grain count of these cells decreased with time. The labeling index of the monocytes of the bone marrow increased during the first 24 hr after in vivo labeling, but during the same period the mean grain counts remained almost constant, with values amounting to about half those of the promonocytes during the first 6 hr of the experiment. The data concerning the labeling indices and the percentage distribution ratio of the promonocytes and monocytes in the bone marrow, and the labeling indices of the peripheral blood monocytes are used to construct a model population. The results lead to the conclusions that the promonocytes are multiplicative cells and that both daughter cells arising from the division of a promonocyte are monocytes. The DNA-synthesis time found for the promonocytes is 13.6 hr. From this value, the average generation time was computed to be 19.5 hr.
Topics: Animals; Autoradiography; Bone Marrow; Bone Marrow Cells; Cell Differentiation; Culture Techniques; DNA; Mice; Mitosis; Monocytes; Thymidine; Tritium
PubMed: 5508379
DOI: 10.1084/jem.132.4.813