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International Journal of Molecular... Apr 2020The intracellular-ubiquitin-ending-enzyme tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is a potent inhibitor of the pro-inflammatory nuclear factor...
The intracellular-ubiquitin-ending-enzyme tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is a potent inhibitor of the pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway. Single nucleotide polymorphisms in TNFAIP3 locus have been associated to autoimmune inflammatory disorders, including Multiple Sclerosis (MS). Previously, we reported a TNFAIP3 down-regulated gene expression level in blood and specifically in monocytes obtained from treatment-naïve MS patients compared to healthy controls (HC). Myeloid cells exert a key role in the pathogenesis of MS. Here we evaluated the effect of specific TNFAIP3 deficiency in myeloid cells including monocytes, monocyte-derived cells (M-MDC) and microglia analyzing lymphoid organs and microglia of mice. TNFAIP3 deletion is induced using conditional knock-out mice for myeloid lineage. Flow-cytometry and histological procedures were applied to assess the immune cell populations of spleen, lymph nodes and bone marrow and microglial cell density in the central nervous system (CNS), respectively. We found that TNFAIP3 deletion in myeloid cells induces a reduction in body weight, a decrease in the number of M-MDC and of common monocyte and granulocyte precursor cells (CMGPs). We also reported that the lack of TNFAIP3 in myeloid cells induces an increase in microglial cell density. The results suggest that TNFAIP3 in myeloid cells critically controls the development of M-MDC in lymphoid organ and of microglia in the CNS.
Topics: Animals; Body Weight; Bone Marrow Cells; Central Nervous System; Female; Flow Cytometry; Granulocyte Precursor Cells; Inflammation; Lymph Nodes; Macrophages; Male; Mice; Mice, Knockout; Microglia; Monocytes; Myeloid Cells; Myelopoiesis; Spleen; Tumor Necrosis Factor alpha-Induced Protein 3
PubMed: 32325694
DOI: 10.3390/ijms21082830 -
Blood Mar 2017Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all--retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common,...
Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all--retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation.
Topics: Blood Coagulation; Cells, Cultured; Chromatin; Endothelial Cells; Fibrin; Fibrinolysis; Granulocyte Precursor Cells; Humans; Leukemia, Promyelocytic, Acute; Tretinoin; Tumor Cells, Cultured
PubMed: 28053193
DOI: 10.1182/blood-2016-09-739334 -
Blood May 2016
Topics: Aged; Antigens, CD; Blood Cell Count; Chromatin; Dyspnea; Fatigue; Granulocyte Precursor Cells; Humans; Leukemia, Myeloid, Acute; Male; Pancytopenia
PubMed: 28092888
DOI: 10.1182/blood-2016-02-698506 -
BMC Genomics Jul 2017RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and...
BACKGROUND
RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. METHODS: Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. RESULTS: This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments.
CONCLUSIONS
These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.
Topics: Gene Library; HL-60 Cells; Humans; RNA, Messenger; Sequence Analysis, RNA; Specimen Handling
PubMed: 28679365
DOI: 10.1186/s12864-017-3900-6 -
American Journal of Hematology Jul 2006Recent studies of acute erythroleukemias have reaffirmed DiGuglielmo's syndrome (M6a, myeloblast-predominant) and disease (M6b, pronormoblast-predominant). M6c (mixed...
Recent studies of acute erythroleukemias have reaffirmed DiGuglielmo's syndrome (M6a, myeloblast-predominant) and disease (M6b, pronormoblast-predominant). M6c (mixed myeloblast/pronormoblast) has also been described. However, MDS is still defined according to the percentage of myeloblasts (% myeloblasts) without including the pronormoblast count. A 20-year retrospective study was performed to identify cases demonstrating >or=50% erythrocytic component and <30% calculated blasts (FAB exclusion criteria) without underlying cause (96 cases). Pronormoblast and myeloblast counts and other variables were analyzed as possible explanatory variables of the variations in survival. Considered alone, increasing % myeloblasts and/or percentage of pronormoblasts (% pronormoblasts) were significant predictors of decreasing survival. When all variables were considered as a multivariate group, the best fitting statistical model for predicting survival was a function of age, % pronormoblasts, IPSS cytopenias, platelet count, and percentage erythrocytic component. Of these, % pronormoblasts was by far the most significant. Nonappearance of % myeloblasts in this model is indicative of high correlations of this count with other variables.
Topics: Age Factors; Disease-Free Survival; Erythroblasts; Erythrocyte Count; Erythrocytes; Granulocyte Precursor Cells; Humans; Leukemia, Erythroblastic, Acute; Male; Middle Aged; Models, Statistical; Multivariate Analysis; Myelodysplastic Syndromes; Platelet Count; Predictive Value of Tests; Prognosis; Retrospective Studies
PubMed: 16755568
DOI: 10.1002/ajh.20563 -
Polish Journal of Pathology : Official... 2022Acute promyelocyte leukaemia (APL) is a subgroup of acute myeloid leukaemia. Dysregulation of clock genes has been revealed to be involved in APL progression. Herein,...
Acute promyelocyte leukaemia (APL) is a subgroup of acute myeloid leukaemia. Dysregulation of clock genes has been revealed to be involved in APL progression. Herein, the mechanism of clock gene aryl hydrocarbon receptor nuclear translocator- like (ARNTL) in APL was explored. The expression of ARNTL, period circadian regulator 1 and 2 (PER1 and PER2) in APL tissue samples and normal samples was analysed by bioinformatic analysis. Gene expression in APL cells was detected by reverse transcription quantitative polymerase chain reaction. Acute promyelocyte leukaemia cell viability and cell cycle progression were assessed by cell counting kit 8 (CCK-8) assays and flow cytometry analyses, respectively. The protein levels of ARNTL and cell cycle markers were examined by western blotting. Interaction between ARNTL and miR-320a/b was confirmed by luciferase reporter assays. Aryl hydrocarbon receptor nuclear translocator-like was overexpressed in marrow tissues of patients with acute myeloid leukaemia and predicted poor outcome. Aryl hydrocarbon receptor nuclear translocator-like knockdown inhibited APL cell viability and arrested APL cells in the G1 phase. Mechanically, ARNTL was targeted by miR-320a/b. Moreover, miR-320a/b upregulation promoted cell cycle arrest in the G1 phase and suppressed the viability of APL cells, and the impacts were reversed by ARNTL overexpression. In conclusion, miR-320a/b suppresses cell viability and leads to cell cycle arrest by suppressing ARNTL in APL.
Topics: Humans; Cell Survival; Aryl Hydrocarbon Receptor Nuclear Translocator; ARNTL Transcription Factors; Granulocyte Precursor Cells; MicroRNAs; Cell Cycle; Leukemia; Cell Proliferation; Cell Line, Tumor
PubMed: 35979756
DOI: 10.5114/pjp.2022.118711 -
JCI Insight Oct 2020Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single...
Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b+CD15+CD10lo/-CD64- band neutrophils and CD66bhiCD15+CD10lo/-CD64+/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation.
Topics: Aged; Antigens, CD; Antigens, Surface; Apoptosis; Autoimmune Diseases; Cell Adhesion Molecules; Cell Line; Cell Lineage; Coculture Techniques; Female; GPI-Linked Proteins; Giant Cell Arteritis; Granulocyte Precursor Cells; Humans; Leukocyte Count; Lewis X Antigen; Male; Middle Aged; Neprilysin; Neutrophils; Oxidation-Reduction; Prognosis; Reactive Oxygen Species; Single-Cell Analysis; Systemic Vasculitis; Temporal Arteries; Vascular Diseases
PubMed: 32960815
DOI: 10.1172/jci.insight.139163 -
International Journal of Molecular... Nov 2019Granulocyte-colony stimulating factor (G-CSF)/nicotinamide phosphoribosyltransferase (NAMPT) signaling has been shown to be crucial for the modulation of neutrophil...
Granulocyte-colony stimulating factor (G-CSF)/nicotinamide phosphoribosyltransferase (NAMPT) signaling has been shown to be crucial for the modulation of neutrophil development and functionality. As this signaling pathway is significantly suppressed by type I interferons (IFNs), we aimed to study how the regulation of neutrophil differentiation and phenotype is altered in IFN-deficient mice during granulopoiesis. The composition of bone marrow granulocyte progenitors and their expression were assessed in bone marrow of type I IFN receptor knockout ( mice and compared to wild-type animals. The impact of NAMPT inhibition on the proliferation, survival, and differentiation of murine bone marrow progenitors, as well as of murine 32D and human HL-60 neutrophil-like cell lines, was estimated. The progressive increase of expression during neutrophil progenitor maturation could be observed, and it was more prominent in IFN-deficient animals. Altered composition of bone marrow progenitors in these mice correlated with the dysregulation of apoptosis and altered differentiation of these cells. We observed that NAMPT is vitally important for survival of early progenitors, while at later stages it delays the differentiation of neutrophils, with moderate effect on their survival. This study shows that IFN-deficiency leads to the elevated NAMPT expression in the bone marrow, which in turn modulates neutrophil development and differentiation, even in the absence of tumor-derived stimuli.
Topics: Animals; Apoptosis; Cell Differentiation; Cell Survival; Granulocyte Colony-Stimulating Factor; Granulocyte Precursor Cells; HL-60 Cells; Humans; Interferons; Mice, Inbred C57BL; Neutrophil Infiltration; Neutrophils; Nicotinamide Phosphoribosyltransferase; Receptor, Interferon alpha-beta; Signal Transduction
PubMed: 31717318
DOI: 10.3390/ijms20225584 -
Haematologica Dec 2019
Topics: Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Cells; Combined Modality Therapy; Erythroid Cells; Follow-Up Studies; Granulocyte Precursor Cells; Humans; Myelodysplastic Syndromes; Prognosis; Retrospective Studies; Stem Cell Transplantation; Survival Rate
PubMed: 31004031
DOI: 10.3324/haematol.2018.212563 -
BMC Biology Nov 2018Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. The nucleus must be stiff enough to withstand cytoskeletal forces, but...
BACKGROUND
Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. The nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through confined spaces, where the nuclear shape must change in order to fit through a constriction. This occurs many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration. Here we characterized the effects of constricted migration in neutrophil-like cells.
RESULTS
Total RNA sequencing identified that migration of neutrophil-like cells through 5- or 14-μm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeletal remodeling. Hi-C was used to capture the genome organization in control and migrated cells. Limited switching was observed between the active (A) and inactive (B) compartments after migration. However, global depletion of short-range contacts was observed following migration with constriction compared to migration without constriction. Regions with disrupted contacts, TADs, and compartments were enriched for inactive chromatin.
CONCLUSION
Short-range genome organization is preferentially altered in inactive chromatin, possibly protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in relation to human health and disease.
Topics: Cell Nucleus; Chromatin; HL-60 Cells; Humans; Neutrophils
PubMed: 30477489
DOI: 10.1186/s12915-018-0608-2