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Haematologica Jul 2022Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/myeloproliferative overlap neoplasm characterized by sustained peripheral blood monocytosis and an...
Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/myeloproliferative overlap neoplasm characterized by sustained peripheral blood monocytosis and an inherent risk for transformation to acute myeloid leukemia (15-30% over 3-5 years). While CMML is morphologically classified into CMML-0, 1 and 2 based on peripheral blood and bone marrow promonocyte/blast counts, a more clinically relevant classification into dysplastic and proliferative subtypes, based on the presenting white blood cell count, is helpful in prognostication and therapeutics. CMML is a neoplasm associated with aging, occurring on the background of clonal hematopoiesis, with TET2 and SRSF2 mutations being early initiating events. The subsequent acquisitions of ASXL1, RUNX1, SF3B1 and DNMT3A mutations usually give rise to dysplastic CMML, while ASXL1, JAK2V617F and RAS pathway mutations give rise to proliferative CMML. Patients with proliferative CMML have a more aggressive course with higher rates of transformation to acute myeloid leukemia. Allogeneic stem cell transplant remains the only potential cure for CMML; however, given the advanced median age at presentation (73 years) and comorbidities, it is an option for only a few affected patients (10%). While DNA methyltransferase inhibitors are approved for the management of CMML, the overall response rates are 40-50%, with true complete remission rates of <20%. These agents seem to be particularly ineffective in proliferative CMML subtypes with RAS mutations, while the TET2mutant/ASXL1wildtype genotype seems to be the best predictor for responses. These agents epigenetically restore hematopoiesis in responding patients without altering mutational allele burdens and progression remains inevitable. Rationally derived personalized/targeted therapies with disease-modifying capabilities are much needed.
Topics: Humans; Leukemia, Myelomonocytic, Chronic; Leukemia, Myelomonocytic, Juvenile; Mutation; Myelodysplastic-Myeloproliferative Diseases; Prognosis
PubMed: 35236051
DOI: 10.3324/haematol.2021.279500 -
American Journal of Hematology Jun 2024Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder with overlapping features of myelodysplastic syndromes and myeloproliferative... (Review)
Review
DISEASE OVERVIEW
Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder with overlapping features of myelodysplastic syndromes and myeloproliferative neoplasms, characterized by prominent monocytosis and an inherent risk for leukemic transformation (~15%-20% over 3-5 years).
DIAGNOSIS
Newly revised diagnostic criteria include sustained (>3 months) peripheral blood (PB) monocytosis (≥0.5 × 10/L; monocytes ≥10% of leukocyte count), consistent bone marrow (BM) morphology, <20% BM or PB blasts (including promonocytes), and cytogenetic or molecular evidence of clonality. Cytogenetic abnormalities occur in ~30% of patients, while >95% harbor somatic mutations: TET2 (~60%), SRSF2 (~50%), ASXL1 (~40%), RAS pathway (~30%), and others. The presence of ASXL1 and DNMT3A mutations and absence of TET2 mutations negatively impact overall survival (ASXL1/TET2 genotype being favorable).
RISK STRATIFICATION
Several risk models serve similar purposes in identifying high-risk patients that are considered for allogeneic stem cell transplant (ASCT) earlier than later. Risk factors in the Mayo Molecular Model (MMM) include presence of truncating ASXL1 mutations, absolute monocyte count >10 × 10/L, hemoglobin <10 g/dL, platelet count <100 × 10/L, and the presence of circulating immature myeloid cells; the resulting 4-tiered risk categorization includes high (≥3 risk factors), intermediate-2 (2 risk factors), intermediate-1 (1 risk factor), and low (no risk factors); the corresponding median survivals were 16, 31, 59, and 97 months. CMML is also classified as being "myeloproliferative (MP-CMML)" or "myelodysplastic (MD-CMML)," based on the presence or absence of leukocyte count of ≥13 × 10/L.
TREATMENT
ASCT is the only treatment modality that secures cure or long-term survival and is appropriate for MMM high/intermediate-2 risk disease. Drug therapy is currently not disease-modifying and includes hydroxyurea and hypomethylating agents; a recent phase-3 study (DACOTA) comparing hydroxyurea and decitabine, in high-risk MP-CMML, showed similar overall survival at 23.1 versus 18.4 months, respectively, despite response rates being higher for decitabine (56% vs. 31%).
UNIQUE DISEASE ASSOCIATIONS
These include systemic inflammatory autoimmune diseases, leukemia cutis and lysozyme-induced nephropathy; the latter requires close monitoring of renal function during leukocytosis and is a potential indication for cytoreductive therapy.
Topics: Humans; Leukemia, Myelomonocytic, Chronic; Risk Assessment; Mutation; Hematopoietic Stem Cell Transplantation
PubMed: 38450850
DOI: 10.1002/ajh.27271 -
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 -
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 -
International Immunopharmacology Jul 2021Chronic obstructive pulmonary disease is characterized by chronic inflammation of the airway and lungs. Accumulating evidence has suggested that erythromycin (EM) plays...
Chronic obstructive pulmonary disease is characterized by chronic inflammation of the airway and lungs. Accumulating evidence has suggested that erythromycin (EM) plays a protective role against cigarette smoke-induced oxidative stress and the inflammatory response. However, the underlying mechanisms remain relatively unclear. The present study aimed to investigate the role of EM in inhibiting cigarette smoke-induced inflammation in human macrophages and its potential mechanism. A Cell Counting Kit-8 assay was used to determine the optimum concentration of EM and cigarette smoke extract (CSE) and it was found that 0.1 and 1% CSE and 0.1, 1.0 and 10 μg/ml EM exerted no significant effect on the cell proliferation activity, whereas 2 and 3% CSE exerted a significant inhibitory effect over the cell proliferation activity. We observed that 10 μmol/ml GW9662 (A PPARγ antagonist) and the presence of 1% CSE could promote the expression and activation of NF-κB p65. And this increased the expression of IL-6, IL-8 and reactive oxygen species (ROS). At the same time, 10 μmol/ml GW9662 and 1% CSE was found to inhibit the expression and activation of peroxisome proliferator activated receptors γ (PPARγ); However, 1 μg/ml EM was discovered to reverse these effects. Co-immunoprecipitation subsequently discovered an interaction between PPARγ and NF-κB p65. In conclusion, the present study suggested that EM may reduce the damage of PPARγ by inhibiting oxidative stress and reducing the expression of ROS and finally relieving cigarette smoke-induced inflammation through the PPARγ/NF-κB signaling pathway in macrophages.
Topics: Cell Proliferation; Erythromycin; Humans; Inflammation; Interleukin-6; Interleukin-8; Macrophages; NF-kappa B; PPAR gamma; Reactive Oxygen Species; Signal Transduction; Smoke; Tobacco Products; Transcription Factor RelA; U937 Cells
PubMed: 34162143
DOI: 10.1016/j.intimp.2021.107775 -
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 -
Frontiers in Immunology 2022Kawasaki disease (KD) is an autoimmune-like vasculitis of childhood involving the coronary arteries. Macrophages require scavenger receptors such as CD36 to effectively...
Kawasaki disease (KD) is an autoimmune-like vasculitis of childhood involving the coronary arteries. Macrophages require scavenger receptors such as CD36 to effectively clear cellular debris and induce self-tolerance. In this study, we hypothesized that CD36 plays an important role in the immunopathogenesis of KD, by aiding in the clearance of plasma mitochondrial DNA, and by amplifying the immune response by activating the inflammasome pathway AIM2. Fifty-two healthy controls, 52 febrile controls, and 102 KD patients were recruited for RT-PCR of target mRNA expression and plasma mitochondrial DNA. Blood samples were obtained 24 hours prior and 21 days after the administration of intravenous immunoglobulin (IVIG) therapy. Patients with acute KD had higher plasma levels of cell-free mitochondrial DNA (ND1, ND4, and COX1), and higher mRNA expressions of CD36 and AIM2 when compared to both healthy and febrile controls. A greater decrease in both CD36 and AIM2 mRNA expression after IVIG therapy was associated with the development of coronary artery lesions. Coronary artery lesions were associated with a larger decrease of CD36 expression following IVIG therapy, which may indicate that prolonged expression of the scavenger receptor may have a protective effect against the development of coronary artery lesions in KD.
Topics: Adolescent; CD36 Antigens; Child; Child, Preschool; Coronary Artery Disease; Coronary Vessels; Female; Gene Expression Profiling; Humans; Infant; Infant, Newborn; Leukocyte Count; Male; Mucocutaneous Lymph Node Syndrome; U937 Cells
PubMed: 35154107
DOI: 10.3389/fimmu.2022.790095 -
Journal of Extracellular Vesicles Apr 2021We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were:...
We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.
Topics: Biomarkers; Cell Line; Chromatography, Gel; Extracellular Vesicles; Humans; Microfluidics; Microscopy, Electron, Transmission; Nanoparticles; Particle Size; Polystyrenes; Single Molecule Imaging; Ultracentrifugation; Ultrafiltration
PubMed: 33850608
DOI: 10.1002/jev2.12079 -
Frontiers in Bioinformatics 2022Osteoclasts are multinucleated cells that exclusively resorb bone matrix proteins and minerals on the bone surface. They differentiate from monocyte/macrophage-lineage...
Osteoclasts are multinucleated cells that exclusively resorb bone matrix proteins and minerals on the bone surface. They differentiate from monocyte/macrophage-lineage cells in the presence of osteoclastogenic cytokines such as the receptor activator of nuclear factor-κB ligand (RANKL) and are stained positive for tartrate-resistant acid phosphatase (TRAP). In vitro, osteoclast formation assays are commonly used to assess the capacity of osteoclast precursor cells for differentiating into osteoclasts wherein the number of TRAP-positive multinucleated cells are counted as osteoclasts. Osteoclasts are manually identified on cell culture dishes by human eyes, which is a labor-intensive process. Moreover, the manual procedure is not objective and result in lack of reproducibility. To accelerate the process and reduce the workload for counting the number of osteoclasts, we developed OC_Finder, a fully automated system for identifying osteoclasts in microscopic images. OC_Finder consists of cell image segmentation with a watershed algorithm and cell classification using deep learning. OC_Finder detected osteoclasts differentiated from wild-type and precursor cells at a 99.4% accuracy for segmentation and at a 98.1% accuracy for classification. The number of osteoclasts classified by OC_Finder was at the same accuracy level with manual counting by a human expert. OC_Finder also showed consistent performance on additional datasets collected with different microscopes with different settings by a different operator. Together, successful development of OC_Finder suggests that deep learning is a useful tool to perform prompt and accurate unbiased classification and detection of specific cell types in microscopic images.
PubMed: 35474753
DOI: 10.3389/fbinf.2022.819570 -
The American Journal of Surgical... Oct 2021Blast evaluation in patients with acute monocytic leukemias (AMoL) is notoriously difficult due to the lack of reliable surface markers and cytologic subtleties on the...
Blast evaluation in patients with acute monocytic leukemias (AMoL) is notoriously difficult due to the lack of reliable surface markers and cytologic subtleties on the aspirate smears. While blasts of most nonmonocytic acute leukemias express CD34, available immunohistochemical antibodies to monocytic blasts also mark normal background mature monocytes. We searched for a potential biomarker candidate by surveying specific gene expression profiles of monocyte progenitors. Our investigations led us to IRF8, which is a lineage-specific transcription factor critical for the production of monocytic and dendritic cell progenitors. In this study, we tested and validated a monoclonal antibody to IRF8 as a novel immunohistochemical stain for trephine core biopsies of human bone marrow. We assessed the expression of IRF8 in 90 cases of AMoL, including posttherapy staging bone marrows, 23 cases of chronic myelomonocytic leukemia, 26 cases of other acute myeloid leukemia subtypes, and 18 normal control marrows. In AMoL, there was high correlation of IRF8-positive cells to aspirate blast count (R=0.95). Comparison of IRF8 staining to aspirate blast percentage in chronic myelomonocytic leukemia also showed good correlation (R=0.86). In contrast, IRF8-positive cells did not correlate with blast count in other subtypes of acute myeloid leukemia (R=0.56) and staining was <5% in all normal control marrows, even those with reactive monocytosis. We found that IRF8 was also weakly reactive in B cells and hematogones, with the latter accounting for rare cases of discrepancies. When IRF8 was used to categorize cases as AMoL, positive for residual leukemia or negative, the sensitivity was 98%, specificity was 82%, positive predictive value was 86%, and negative predictive value was 98%. These results demonstrate that IRF8 may serve as a clinically useful immunostain to diagnose and track AMoLs on bone marrow core biopsies. This can be particularly impactful in the setting of poor aspiration and focal blast increase. In the era of new targeted therapies that have been reported to induce monocytic outgrowths of leukemia, a marker for malignant monoblasts may prove even more critical.
Topics: Aged; Biomarkers, Tumor; Biopsy; Bone Marrow Examination; Female; Humans; Immunohistochemistry; Interferon Regulatory Factors; Leukemia, Monocytic, Acute; Male; Middle Aged; Monocyte-Macrophage Precursor Cells; Predictive Value of Tests; Proof of Concept Study; Reproducibility of Results
PubMed: 34172624
DOI: 10.1097/PAS.0000000000001765