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Immunopharmacology and Immunotoxicology Jun 2020Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and joint destruction. Excessive proliferation of fibroblast-like synoviocytes...
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and joint destruction. Excessive proliferation of fibroblast-like synoviocytes (FLS) and over-expression of angiogenic factors play a crucial role in pannus formation and joint destruction in RA. Clarification of the role of cholinergic agonists in modulation of inflammation and immune system reactions is progressively ongoing. In this study, the anti-angiogenic effect of two cholinergic agonists, nicotine and ARR17779, on human FLS, and monocytic cell lines (U937) was evaluated. The cells were cultured in DMEM supplemented with 10% FBS and treated with different doses of nicotine and ARR17779 in the presence of TNF-α, LPS, and IFN-γ. After 48 h, cell number was counted in different groups. After RNA extraction, cDNA was synthesized and the expression of VEGF and MMPs has been evaluated by real-time PCR using specific primers and probes. VEGF was assayed in U937 cell line supernatant using ELISA method. Both nicotine and ARR17779 inhibited FLS and U937 cell proliferation. Cholinergic agonists reduced the expression of MMPs and VEGF. VEGF level in supernatant of U937 cells treated with cholinergic agonists was also reduced. Our results suggest that cholinergic agonists can modulate pathological conditions related to pannus formation in conditions. Based on these results, cholinergic agonists can be considered as novel therapeutic options in RA. Further animal studies are needed before introducing these agents into clinical uses.
Topics: Cell Culture Techniques; Cell Proliferation; Cholinergic Agonists; Cytokines; Dose-Response Relationship, Drug; Fibroblasts; Gene Expression; Humans; Matrix Metalloproteinases; Monocytes; Synoviocytes; U937 Cells; Vascular Endothelial Growth Factor A; alpha7 Nicotinic Acetylcholine Receptor
PubMed: 32248717
DOI: 10.1080/08923973.2020.1745830 -
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 -
Annales de Biologie Clinique Oct 2019The discovery of a monocytosis is a frequent phenomenon, requiring confirmation by reading under a microscope by an experimented biologist, to overcome usual cytological... (Review)
Review
The discovery of a monocytosis is a frequent phenomenon, requiring confirmation by reading under a microscope by an experimented biologist, to overcome usual cytological traps such as the presence of hairy cells, promonocytes or monoblasts. In the vast majority of cases the secondary origin is very easily found by the context and/or the presence of a biological inflammatory syndrome. More rarely the diagnosis is directed towards an eosinophilic pathology or an acute leukemia. In other cases, CMML, MPN or MDS with monocytosis may be highlighted. In the absence of any pathognomonic element and the presence of "borderline" forms the differential diagnosis between these 3 entities is not always straightforward, requiring, according to WHO, molecular investigations and elimination of any reactive cause of monocytosis. Although histological, immunohistochemical and phenotypic flow cytometric studies are not currently recommended by WHO, these investigations could be of interest in the evaluation of difficult cases.
Topics: Adult; Age of Onset; Algorithms; Clinical Laboratory Techniques; Diagnosis, Differential; Humans; Leukocyte Count; Monocytes; Myelodysplastic Syndromes
PubMed: 31486402
DOI: 10.1684/abc.2019.1475 -
Journal of Extracellular Vesicles 2019Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly,...
Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum "extra-depletion" protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood.
PubMed: 31303981
DOI: 10.1080/20013078.2019.1628592 -
Journal of Microbiology (Seoul, Korea) Aug 2019Low-density lipoprotein (LDL) was recently reported to be an opsonin, enhancing the phagocytosis of group A Streptococcus (GAS) by human monocytic leukemia U937 cells...
Low-density lipoprotein (LDL) was recently reported to be an opsonin, enhancing the phagocytosis of group A Streptococcus (GAS) by human monocytic leukemia U937 cells due to the binding of LDL to some GAS strains. We postulated that LDL might also promote the opsonophagocytosis of Pseudomonas aeruginosa by U937 cells since this bacterium interacts with LDL. In this study, P. aeruginosa (CMCC10104), U937 cells, and human LDL were used in phagocytosis assays to test our hypothesis. Escherichia coli strain BL21, which does not interact with LDL, was used as a negative control. Colony counting and fluorescence microscopy were used to determine the bacterial quantity in the opsonophagocytosis assays. After incubation of U937 cells and P. aeruginosa with LDL (100 µg/ml) for 15 and 30 min, phagocytosis was observed to be increased by 22.71% and 32.90%, respectively, compared to that seen in the LDL-free group. However, LDL did not increase the phagocytosis of E. coli by U937 cells. In addition, we identified CD36 as a major opsonin receptor on U937 cells, since an anti-CD36 monoclonal antibody, but not an anti-CD4 monoclonal antibody, almost completely abolished the opsonophagocytosis of P. aeruginosa by U937 cells.
Topics: Antibodies, Monoclonal; Escherichia coli; Humans; Lipoproteins, LDL; Opsonin Proteins; Phagocytosis; Pseudomonas aeruginosa; U937 Cells
PubMed: 31089970
DOI: 10.1007/s12275-019-8413-3 -
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 -
European Review For Medical and... Apr 2019The aim of this study was to elucidate the biological function of long non-coding RNA (lncRNA) HOTTIP (HOXA transcript at the distal tip) in the development of acute...
OBJECTIVE
The aim of this study was to elucidate the biological function of long non-coding RNA (lncRNA) HOTTIP (HOXA transcript at the distal tip) in the development of acute myeloid leukemia (AML), and to investigate the potential mechanism.
PATIENTS AND METHODS
Relative expression levels of HOTTIP, microRNA-608 and DDA1 in AML patients were determined by quantitative Real-time polymerase chain reaction (qRT-PCR). Meanwhile, the expressions of these genes in AML cell lines were detected as well. The regulatory effects of HOTTIP, microRNA-608 and DDA1 on the proliferative ability and cell cycle progression of AML cells were examined by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Dual-luciferase reporter gene assay was performed to confirm the binding condition of microRNA-608 to HOTTIP and DDA1. Finally, the specific role of HOTTIP/microRNA-608/DDA1 axis in the development of AML was verified through a series of rescue experiments.
RESULTS
HOTTIP was highly expressed in AML-M5 patients than normal controls. No significant difference in HOTTIP expression was found between patients with other subtypes of AML (M0, M1, M2, M3, M4 and M6) and normal controls. HOTTIP expression was significantly up-regulated in AML cell lines U-937 and THP-1. Up-regulation of HOTTIP remarkably promoted the proliferative potential and cell cycle progression of AML cells. Dual-luciferase reporter gene indicated that HOTTIP could bind to microRNA-608, which was lowly expressed in AML-M5 patients. Overexpression of microRNA-608 significantly inhibited the proliferative ability and cell cycle progression of U-937 and THP-1 cells. More importantly, microRNA-608 could partially reverse the regulatory effect of HOTTIP on AML cells. Meanwhile, DDA1 was verified as the target of microRNA-608. Subsequent experiments elucidated that DDA1 significantly accelerated the proliferation and cell cycle of AML cells. Furthermore, DDA1 could reverse the inhibitory effect of microRNA-608 on proliferative ability and cell cycle progression of AML cells.
CONCLUSIONS
HOTTIP accelerated the proliferative ability and cell cycle of AML cells via up-regulating DDA1 expression by sponging microRNA-608.
Topics: Cell Cycle; Cell Proliferation; DNA-Binding Proteins; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; MicroRNAs; RNA, Long Noncoding; U937 Cells
PubMed: 31002141
DOI: 10.26355/eurrev_201904_17569 -
Experimental Parasitology Feb 2019The intraerythrocytic malaria parasite digests haemoglobin to provide amino acids for metabolism and releases toxic haem that is sequestered into haemozoin, a non-toxic,...
The intraerythrocytic malaria parasite digests haemoglobin to provide amino acids for metabolism and releases toxic haem that is sequestered into haemozoin, a non-toxic, insoluble, crystalline pigment. Following erythrocyte rupture, haemozoin is released into circulation and phagocytosed by monocytes. Phagocytosed haemozoin and antimalarial drugs have both been reported to modulate monocyte functions. This study determined the effects of therapeutic concentrations of seven antimalarial drugs; amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of β-haematin (synthetic haemozoin) by two monocytic cell lines, J774A.1 and U937, and human peripheral blood mononuclear cells. A novel spectrophotometric method based on the absorbance (O.D 400 nm) of alkali/SDS treated monocytes containing β-haematin was developed to complement counting phagocytosis with microscopy. The method has potential use for the large scale screening of monocyte phagocytic activity. Artemisinin, quinine, primaquine and pyrimethamine activated β-haematin phagocytosis by 12% or more, whereas amodiaquine, chloroquine and doxycyline inhibited β-haematin phagocytosis. In contrast, antimalarial drugs had minimal inhibitory effects on the phagocytosis of latex beads with only quinine resulting in more than 20% inhibition. Antimalarial drugs appear to alter monocyte phagocytic activity which has implications for the treatment, pathogenicity and adjunct therapies for malaria.
Topics: Amodiaquine; Animals; Antimalarials; Artemisinins; Cell Count; Cell Line; Chloroquine; Doxycycline; Electron Probe Microanalysis; Heme; Hemeproteins; Humans; Leukocytes, Mononuclear; Mice; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Monocytes; Peroxidase; Phagocytosis; Primaquine; Pyrimethamine; Quinine; Spectrophotometry; Temperature; U937 Cells
PubMed: 30562480
DOI: 10.1016/j.exppara.2018.12.002 -
Journal of Cellular Biochemistry Mar 2019Dysregulation of microRNAs is closely implicated in the initiation and progression of human cancers including acute myeloid leukemia (AML). Though miR-139-5p was...
Dysregulation of microRNAs is closely implicated in the initiation and progression of human cancers including acute myeloid leukemia (AML). Though miR-139-5p was reported to be a potent tumor suppressor in adult AML, its underlying molecular mechanism in AML remains to be further defined. Herein, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were conducted to determine the expressions of miR-139-5p and tetraspanin3 (Tspan3) in AML patients and cells. Luciferase reporter assay, qRT-PCR, and Western blot analysis were carried out to detect the interaction between miR-139-5p and Tspan3. Cell proliferation, cell cycle distribution, invasion, and migration were evaluated by cell counting kit-8, flow cytometry, transwell invasion, and migration assays, respectively. Western blot analysis was conducted to determine phosphorylated-protein kinase B (Akt) and Akt levels. We found that a significant reduction in miR-139-5p expression and a prominent increase in Tspan3 expression were observed in AML patients and cells. Tspan3 was confirmed as a direct target of miR-139-5p and was negatively modulated by miR-139-5p. Rescue experiments showed that overexpression of miR-139-5p constrained cell proliferation, invasion and migration capabilities, and induced cell cycle arrest at the S phase in AML cells, which were partially reversed by Tspan3 overexpression. In addition, we found that miR-139-5p suppressed the phosphoinositide 3-kinase (PI3K)/Akt pathway in AML cells by targeting Tspan3. In conclusion, our study concluded that miR-139-5p suppressed the leukemogenesis in AML cells by targeting Tspan3 through inactivation of the PI3K/Akt pathway, providing a better understanding of AML progression.
Topics: Cell Proliferation; Genes, Tumor Suppressor; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; MicroRNAs; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Neoplasm; Signal Transduction; THP-1 Cells; Tetraspanins; U937 Cells
PubMed: 30367526
DOI: 10.1002/jcb.27728 -
Cytometry. Part B, Clinical Cytometry Nov 2018The purpose of this study was to determine whether immunophenotypic profiles detected by flow cytometry are useful in differentiating chronic myelomonocytic leukemia...
OBJECTIVES
The purpose of this study was to determine whether immunophenotypic profiles detected by flow cytometry are useful in differentiating chronic myelomonocytic leukemia (CMML) from reactive monocytosis, and between CMML subtypes.
METHODS
Eight-color flow cytometry was used to immunophenotype blasts, monocytes, and granulocytes in the bone marrow of 34 patients with CMML and 12 patients with reactive monocytosis.
RESULTS
Bone marrow myeloblast, promonocyte, and monocyte counts by flow cytometry were significantly higher in the CMML group than in the reactive monocytosis group. Myeloblast aberrancies were present in all CMML patients as compared with 2 of 12 (16.7%) reactive monocytosis patients (P < 0.001). The number of blast aberrancies ranged from one to nine (median, four) in CMML patients and 94.1% of CMML cases exhibited ≥ two aberrancies. In contrast, two reactive monocytosis cases showed only one phenotypic abnormality of blasts. Monocyte and granulocyte aberrancies were present in 26 of 34 (76.5%) and in 31 of 34 (91.2%) CMML patients, respectively. Decreased side scatter (SSC) and abnormal CD11b/CD13/CD16 maturation pattern in granulocytes were more frequent in CMML than in reactive monocytosis. No significant differences in antigen expression were detected between the CMML subtypes except that altered CD45/SSC pattern on the blasts was more commonly observed in CMML-0/1 than in CMML-2.
CONCLUSIONS
CMML has phenotypic aberrancies in monocytes, granulocytes, and more frequently in myeloblasts. Aberrant expression of two or more antigens in myeloblasts by flow cytometry has a high sensitivity (94.1%) and a high specificity (100%) to differentiate CMML from reactive monocytosis. © 2018 International Clinical Cytometry Society.
Topics: Adult; Aged; Aged, 80 and over; Female; Flow Cytometry; Humans; Immunophenotyping; Leukemia, Myelomonocytic, Chronic; Male; Middle Aged; Monocytes; Young Adult
PubMed: 30334354
DOI: 10.1002/cyto.b.21721