The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils of polycythemia vera.

BACKGROUND: The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes. DESIGN AND METHODS: We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63(+) basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor. RESULTS: We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63(+) basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63(+) basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden. CONCLUSIONS: These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells’ abnormal function and, putatively, in the pathogenesis of pruritus

Megakaryocyte contribution to bone marrow fibrosis: many arrows in the quiver

In Primary Myelofibrosis (PMF), megakaryocyte dysplasia/hyperplasia determines the release of inflammatory cytokines that, in turn, stimulate stromal cells and induce bone marrow fibrosis. The pathogenic mechanism and the cells responsible for progression to bone marrow fibrosis in PMF are not completely understood. This review article aims to provide an overview of the crucial role of megakaryocytes in myelofibrosis by discussing the role and the altered secretion of megakaryocyte-derived soluble factors, enzymes and extracellular matrices that are known to induce bone marrow fibrosis. Additionally, we describe recent evidences showing that the role of megakaryocytes in tissue fibrosis is not limited to the bone marrow

Dexamethasone Predisposes Human Erythroblasts Toward Impaired Lipid Metabolism and Renders Their ex vivo Expansion Highly Dependent on Plasma Lipoproteins

Cultures of stem cells from discarded sources supplemented with dexamethasone, a synthetic glucocorticoid receptor agonist, generate cultured red blood cells (cRBCs) in numbers sufficient for transfusion. According to the literature, however, erythroblasts generated with dexamethasone exhibit low enucleation rates giving rise to cRBCs that survive poorly in vivo. The knowledge that the glucocorticoid receptor regulates lipid metabolism and that lipid composition dictates the fragility of the plasma membrane suggests that insufficient lipid bioavailability restrains generation of cRBCs. To test this hypothesis, we first compared the expression profiling of erythroblasts generated with or without dexamethasone. This analysis revealed differences in expression of 55 genes, 6 of which encoding proteins involved in lipid metabolism. These were represented by genes encoding the mitochondrial proteins 3-Hydroxymethyl-3-Methylglutaryl-CoA lyase, upregulated, and 3-Oxoacid CoA-Transferase1 and glycerol-3-phosphate acyltransferase1, both downregulated, and the proteins ATP-binding cassette transporter1 and Hydroxysteroid-17-Beta-Dehydrogenase7, upregulated, and cAMP-dependent protein kinase catalytic subunit beta, downregulated. This profiling predicts that dexamethasone, possibly by interfering with mitochondrial functions, impairs the intrinsic lipid metabolism making the synthesis of the plasma membrane of erythroid cells depend on lipid-uptake from external sources. Optical and electron microscopy analyses confirmed that the mitochondria of erythroblasts generated with dexamethasone are abnormal and that their plasma membranes present pebbles associated with membrane ruptures releasing exosomes and micro-vesicles. These results indicate that the lipid supplements of media currently available are not adequate for cRBCs. To identify better lipid supplements, we determined the number of erythroblasts generated in synthetic media supplemented with either currently used liposomes or with lipoproteins purified from human plasma [the total lipoprotein fraction (TL) or its high (HDL), low (LDL) and very low (VLDL) density lipoprotein components]. Both LDL and VLDL generated numbers of erythroid cells 3-2-fold greater than that observed in controls. These greater numbers were associated with 2–3-fold greater amplification of erythroid cells due both to increased proliferation and to resistance to stress-induced death. In conclusion, dexamethasone impairs lipid metabolism making ex vivo expansion of erythroid cells highly dependent on lipid absorbed from external sources and the use of LDL and VLDL as lipid supplements improves the generation of cRBCs

Hepatic Lysosomal Acid Lipase and lipophagy in the progression of NAFLD

Lysosomal Acid Lipase (LAL) is an acidic enzyme that degrades cholesterol-ester and triglyceride inside lysosomes. Both genetic LAL deficiency and non-alcoholic fatty liver disease (NAFLD) are featured by lipid accumulation in hepatocyte leading to steatosis and eventually liver failure. Recently, a deficit in blood LAL activity was found in NAFLD patient (1). Lipophagy plays a pivotal role in degradation of lipids in the liver and consists in autophagic sequestration of lipid droplets and their degradation inside lysosomes by LAL (2). p62 serves as an autophagy/lipophagy receptor for selective autophagy and accumulates when the autophagy is blocked. We aimed to evaluate the hepatic expression of LAL in NAFLD patients and healthy subjects and to verify its association with histopathological features. Furthermore, we aimed to compare LAL levels with autophagic flux and lysosomal compartment status (LAMP1-positive vesicles). LAL expression was reduced in NAFLD patients with respect to healthy subjects (

E-cigarettes fluids trigger molecular and morphological response in oral fibroblasts

Electronic-cigarettes (e-cigarettes) have been recently advertised as a safe alternative to the traditional ones and a possible smoking cessation tool. This electronic device was designed to transform a solution of variable compounds (some of them approved as food additives), in an inhalable aerosol. However, their safety is still not fully know (Lerner et al. 2016). The cytotoxicity of the fluids on human gingival fibroblasts (HGFs) was demonstrated on a previous study by Sancilio et al. (2016) where the occurrence of oxidative stress and apoptosis was found following the exposure to nicotine containing fluids. The aim of this study was to investigate the HGF biological response to e-cigarettes liquids (with and without nicotine) and to clarify the molecular mechanisms driving the cytotoxicity exerted by fluids themselves. To this purpose, cells were treated with e-cigarette fluids containing nicotine (final concentration 1mg/mL) and the equivalent volume of a fluid without nicotine, for times up to 48 h. Lactate Dehydrogenase Assay (LDH), electronic microscopy analysis, collagen I production, flow cytometry lysosome compartment evaluation and western blotting LC3 (microtubule-associated protein 1A/1B-light chain 3) expression were performed. Fluids containing nicotine exerted cytotoxicity as demonstrated by the increased levels of LDH, in parallel to the formation of numerous vacuoles in the cytoplasm, as well as a decrease in collagen I production and an augmented LC3 II expression which characterized autophagy occurrence In conclusion E-cigarette fluids (with and without nicotine) trigger modification ultrastructure, collagen production and lysosomal compartment in HGFs, suggesting an involvement in the pathogenesis of oral diseases

TGF-beta bioavailability is increased by a new interaction between megakaryocytes and fibrocytes activated in the Gata 1 low mouse

Primary myelofibrosis is the most severe of the Philadelphia-negative myeloproliferative neoplasms and is associated with progressive TGF-β1-dependent scaring of the hematopoietic microenvironment which causes hematopoietic failure in the spleen.Nevertheless, the pathogenetic roleof TGF beta is still unclear because of the modest (2-fold) increases in its plasma levels, both in patients and in animal models. Transmission electron-microscopy (TEM) observations identified that spleen from PMF patients and Gata1low mice contained megakaryocytes with abnormally high levels of TGF-β and collagen fibres embedded in their cytoplasm. Additional immuno-TEM observations of spleen from Gata1low mice revealed the presence of numerous activated fibrocytes establishing with their protrusions a novel cellular interaction, defined as peripolesis, with megakaryocytes. These protrusions infiltrated the megakaryocyte cytoplasm releasing collagen that was eventually detected in its mature polymerized form. Megakaryocytes, engulfed with mature collagen fibres, acquired the morphology of para-apoptotic cells and, in the most advanced cases, were recognized as polylobated heterochromatic nuclei surrounded by collagen fibres strictly associated with TGF-β. These areas contained concentrations of TGF-β-gold particles ~1000-fold greater than normal and numerous myofibroblasts, an indication that TGF-β was bioactive. Loss-of-function studies indicated that peripolesis between megakaryocytes and fibrocytes required both TGF-β, possibly for inducing fibrocyte activation, and P-selectin, possibly for mediating interaction between the two cell types. Loss-of-function of TGF-β and P-selectin also prevented fibrosis. These observations identify that myelofibrosis is associated with pathological increases of TGF-β bioavailability and suggest a novel megakaryocyte-mediated mechanism that may increase TGF-β bioavailability in chronic inflammation

Molecular and histological traits of reduced lysosomal acid lipase activity in the fatty liver

Recent studies demonstrated reduced blood lysosomal acid lipase (LAL) activity in patients with nonalcoholic fatty liver disease (NAFLD). We aimed to verify hepatic LAL protein content and activity in in vitro and in vivo models of fat overload and in NAFLD patients. LAL protein content and activity were firstly evaluated in Huh7 cells exposed to high-glucose/high-lipid (HGHL) medium and in the liver of C57BL/6 mice fed with high-fat diet (HFD) for 4 and 8 months. LAL protein was also evaluated by immunohistochemistry in liver biopsies from 87 NAFLD patients and 10 controls, and correlated with hepatic histology. Huh7 cells treated with HGHL medium showed a significant reduction of LAL activity, which was consistent with reduced LAL protein levels by western blotting using an antibody towards the N-term of the enzyme. Conversely, antibodies towards the C-term of the enzyme evidenced LAL accumulation, suggesting a post-translational modification that masks the LAL N-term epitope and affects enzymatic activity. Indeed, we found a high rate of ubiquitination and extra-lysosomal localization of LAL protein in cells treated with HGHL medium. Consistent with these findings, inhibition of proteasome triggered dysfunctional LAL accumulation and affected LAL activity. Accumulation of ubiquitinated/dysfunctional LAL was also found in the liver of HFD fed mice. In NAFLD patients, hepatic levels of non-ubiquitinated/functional LAL were lower than in controls and inversely correlated with disease activity and some of the hallmarks of reduced LAL. Fat overload leads to LAL ubiquitination and impairs its function, possibly reducing hepatic fat disposal and promoting NAFLD activity