A1AT In Human Neutrophil Granulocytes.

Abstract Abstract 3784 Alpha-1-antitrypsin (A1AT) is an important inhibitor of the neutrophil serine proteases elastase, cathepsin G, and proteinase 3. A1AT is produced mainly by the liver and secreted to plasma. A1AT deficiency caused by the PiZZ mutation in the A1AT gene leads to accumulation of mutated A1AT in the liver which may induce liver cell necrosis and necessitate liver transplantation. In a recently performed profiling of mRNA expression during terminal neutrophil differentiation in the bone marrow, we found that A1AT mRNA increases from the promyelocyte stage and up, indicating that A1AT is a constituent of all neutrophil granules. We examined the localization and production of A1AT in healthy donor neutrophils and investigated whether the structure or function of neutrophils is affected in individuals with A1AT deficiency. RT- PCR for A1AT performed on neutrophil precursors isolated from normal human bone marrow showed that the mRNA level is highly upregulated as the cells mature in the bone marrow and even increases further as the cells enter the blood stream. Biosynthesis studies revealed that A1AT is produced by all stages of neutrophil maturation in the bone marrow and is efficiently retained in the cells as judged by pulse chase studies. Neutrophils from circulating blood also produce A1AT but this is not retained in the cells. Stimulation of neutrophils from peripheral blood with G-CSF during 24 hours resulted in a 20 fold increase in A1AT biosynthesis which was largely released to the medium. Subcellullar fractionation of blood neutrophils on a 4-layer Percoll density gradient revealed 3 forms of A1AT. A doublet band at 37 and 44 kD both with immunoreactivity against A1AT was observed in fractions corresponding to azurophil granules (cf biosynthesis of this form in promyelocytes). A band with mw of 52 kD, corresponding to the form present in blood plasma, was observed in fractions that contain NGAL, a marker of specific granules and in fractions that contain gelatinase. The 52 kD band was also observed in fractions containing albumin as expected, since secretory vesicles contain plasma proteins. The localization of A1at in neutrophil granules was further confirmed by exocytosis studies. Neutrophils were stimulated with PMA which mobilizes secretory vesicles and gelatinase granules efficiently and approximately 50% of specific granules. Only the 52 mw form of A1AT was released from cells during stimulation while none of the 37/44 double band was released. This is in agreement with localization of this double band in azurophil granules and with localization of the 52 kD form in specific granules, gelatinase granules and secretory vesicles. In addition, a high molecular weight form of A1AT was observed at 76 kD corresponding to the mw of A1AT complexed with neutrophil elastase. We isolated neutrophils from patients with the ZZ genotype of A1AT deficiency which had either been liver transplanted or lung transplanted. The neutrophils were examined by electron microscopy for detection of structural abnormalities and by exocytosis studies for detection of functional abnormalities. Electron micrographs did not reveal any abnormality in neutrophil structure and in none of the neutrophils examined (from 6 patients) did we observe abnormal granules akin to the intracellular accumulation of A1AT in liver cells from patients. We did, however, observe reduction in the total intracellular amount of A1AT in neutrophils from patients that had been lung transplanted but not in neutrophils from liver transplanted patients. This most likely reflects that secretory vesicles of neutrophils from lung transplanted will not contain A1AT as this is still severely deficient in plasma from lung transplanted patients, while liver transplanted patients will have normal levels of A1AT in plasma and hence take up normal amounts into their secretory vesicles. Release of granule proteins in response to stimulation by fMLP or PMA did not reveal any functional abnormality in neutrophils from A1AT deficient patients. Based on these studies we conclude that the A1AT deficiency does not inflict functional or structural abnormalities on neutrophils, and suggest that A1AT generated and released from neutrophils may contribute to anti-protease defense in tissues. Disclosures: No relevant conflicts of interest to declare.

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Immunomodulatory Derivatives of Thalidomide (IMiD)-Induced Neutropenia Is Associated with PU.1 Downregulation and Myeloid Maturation Arrest

Blood ◽

10.1182/blood.v112.11.845.845 ◽

2008 ◽

Vol 112 (11) ◽

pp. 845-845 ◽

Cited By ~ 1

Author(s):

Rekha Pal ◽

G. David Roodman ◽

Markus Mapara ◽

Lynn Mocsinski ◽

Alan F List ◽

...

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Human Subjects ◽

Cathepsin G ◽

Severe Neutropenia ◽

Neutrophil Granulocytes ◽

Maturation Arrest ◽

Human Cd34 ◽

Derivatives Of

Abstract Previous clinical trials have shown that immunomodulatory derivatives of thalidomde (IMiDs) including lenalidomide or pomalidomide induce high response rates in patients with relapsed or refractory multiple myeloma. However, the most dose limiting adverse effect of IMiDs is severe neutropenia. This is in contrast to our findings that IMiDs promote a shift in hematopoietic progenitor lineage commitment toward myeloid precursors with a subsequent maturation stop. In colony forming assays using human CD34+ hematopoietic precursors treated with IMiDs, we observed a highly significant increase in myeloid progenitor formation at the expense of erythroid colonies. Treatment of hematopoietic progenitors with IMiDs showed an upregulation of CD33, a marker for immature myeloid cells. Further, in vitro treatment with IMiDs resulted in downregulation of the transcription factor PU.1. PU.1 gene knock-down models show increased granulopoiesis with impaired neutrophil maturation, resulting in an accumulation of promyelocytes. In accordance with that, Cathepsin G as a promyelocytic marker was highly upregulated under treatment with IMiDs measured by high-density oligonucleotide microarray, quantitative RT-PCR, western blot and ELISA, confirming the maturation arrest of neutrophil granulocytes. To evaluate medullary changes in granulopoiesis in human subjects treated with lenalidomide, we evaluated sequential bone marrows from 6 patients comparing bone marrow features prior to treatment, during treatment, and at the time of grade 4 neutropenia. Marrows at the time of neutropenia showed an accumulation of myeloid precursors (myelocytes, metamyelocytes) supporting impaired myeloid maturation. In 5 out of 6 patients the myeloid:erythroid (M:E) ratio increased with an M:E median of 1.95 at pretreatment and 9.2 at the time of the neutrophil nadir. At the same time bone marrow cellularity was unchanged. In summary we show that IMiDs downregulate PU.1, a key transcription factor involved in granulocyte differentiation. The loss of PU.1 results in medullary accumulation of immature myeloid precursors, with corresponding neutropenia in the peripheral blood. Further high amounts of azurophilic granules of promyelocytes subsequently increase Cathepsin G and neutrophil elastase 2 resulting in enhanced platelet aggregation and a possible higher risk for venous thromboembolism. Our findings show that IMiDs do not induce neutropenia by marrow suppression but rather by transient block of maturation, which might be overcome by application of G-CSF.

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Induced Pluripotent Stem Cell-Derived Neutrophil Granulocytes - Flow Cytometry Analysis Reveals Distinct Subpopulations

Blood ◽

10.1182/blood-2021-150495 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3126-3126

Author(s):

Stephanie Frenz ◽

Raffaele Conca ◽

Sebastian Hesse ◽

Monika I. Linder ◽

Christoph Klein

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Single Cell ◽

Conflicts Of Interest ◽

Neutrophil Granulocytes ◽

Mesoderm Induction ◽

Health And Disease ◽

Induced Pluripotent ◽

Neutrophil Differentiation

Abstract Severe congenital neutropenia (SCN) comprises a spectrum of monogenic disorders characterized by impaired differentiation and function of neutrophil granulocytes. Since animal models often do not fully recapitulate human SCN phenotypes and primary bone marrow samples of patients are scarce, alternative strategies are desirable to study the genetic causes and mechanisms. Induced pluripotent stem cells (iPSCs) can be differentiated into neutrophil granulocytes, thereby presenting an excellent tool to study hematopoiesis and especially neutrophil differentiation in health and disease in vitro. Recently, neutrophil progenitors and mature neutrophils of murine and human bone marrow have been characterized by single-cell RNA sequencing, mass cytometry and flow cytometry and are now referred to as proNeu, preNeu, immature-Neu and mature-Neu, at least in part reflecting conventional morphological classification of myeloblasts, promyelocytes, myelocytes/metamyelocytes and band and segmented neutrophils, respectively. Here we developed a flow cytometry antibody panel and gating strategy, which robustly identified distinct myeloid subsets in iPS-derived neutrophils. We adopted a differentiation protocol, which consists of feeder- and serum-free differentiation of iPS cells by mesoderm induction and patterning, followed by lineage progression through hemogenic endothelium to hematopoietic progenitors and finally mature neutrophil granulocytes. Floating cells arise, which can be harvested continuously and analyzed by flow cytometry. Based on expression of cell surface molecules, we defined four subpopulations: After selecting for single, live, CD45 + and CD14 - cells, the different progenitor stages were first defined by their expression of CD117 and CD49d. CD117 midCD49d high cells were further stratified into SSC lowCD34 + cells and SSC highCD34 - cells, representing myeloblasts (proNeu1) and promyelocytes (proNeu2/preNeu), respectively. These cells progressed to CD117 -CD49d mid and were CD11b +CD101 +, which defines myelocytes/metamyelocytes (immature-Neus). CD117 -CD49d low cells were CD11b +CD101 + and expressed additionally CD16, resembling band/segmented neutrophils (mature-Neus). Additionally, these iPS-derived cells progressively expressed CD35, which is also a maturation marker of human myeloid cells in vivo. May-Grünwald-Giemsa staining of these four subpopulations (CD117 midCD49d highSSC lowCD34 +, CD117 mid CD49d highSSC highCD34 -, CD117 - CD49d midand CD117 - CD49d low) revealed homogenous populations of sorted cells, morphologically resembling myeloblasts, promyelocytes, myelocytes/metamyelocytes and band/segmented neutrophils, respectively. Ongoing studies in our lab make use of this model to a) validate the functional significance of rare genetic variants and b) further assess transcriptomic and proteomic changes on a single cell level. Thus, we provide a promising tool to study neutrophil differentiation in health and disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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A Novel Observation That Heme Oxygenase-1 (HO-1) Deficient Mice Are Easy Mobilizers and That HO-1 Plays An Important Role in Maintaining Expression of SDF-1 in Bone Marrow (BM) Stroma and Promotes Retention of Hematopoietic Stem/Progenitor Cells (HSPCs) in the Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v118.21.315.315 ◽

2011 ◽

Vol 118 (21) ◽

pp. 315-315

Author(s):

Marcin Wysoczynski ◽

Janina Ratajczak ◽

Gregg Rokosh ◽

Roberto Bolli ◽

Mariusz Z Ratajczak

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Heme Oxygenase ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Mrna Level ◽

Heme Oxygenase 1 ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Cell Counts

Abstract Abstract 315 Background. Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. Two distinct HO isoforms have been identified: HO-2, which is constitutively expressed, and HO-1, which is stress-responsive and plays an important function in various physiological and pathophysiological states associated with cellular stress. HO-1 plays a role in ischemic/reperfusion injury, atherosclerosis, and cancer. It has also been reported that HO-1 regulates expression of a-chemokine stromal derived factor-1 (SDF-1) in myocardium (J Mol Cell Cardiol.2008;45:44–55). Aim of study. Since SDF-1 plays a crucial role in retention and survival of hematopoietic stem cell/progenitor cells (HSPCs) in BM, we become interested in whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach. To address this issue, we employed several complementary strategies to investigate HO-1−/−, HO+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) chemotactic responsiveness of HSPCs to an SDF-1 gradient, iv) adhesiveness of clonogenic progenitors, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100 assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. Results: Our data indicate that under normal, steady-state conditions, HO-1−/− and HO+/– mice have normal peripheral blood cell counts and numbers of circulating CFU-GM. Interestingly, lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. Next, BMMNCs from HO-1−/−have normal expression of the SDF-1-binding receptor, CXCR4, but a 5-times lower level of CXCR7, which is another SDF-1-binding receptor. Of note, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1−/−animals responded more robustly to an SDF-1 gradient, and HO-1−/−animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into peripheral blood in response to G-CSF and AMD3100. Conclusions: Our data demonstrate for the first time that heme oxygenase plays an important and underappreciated role in BM retention of HSPCs and may affect their trafficking. Since small non-toxic molecular inhibitors of HO-1 have been developed for clinical use (e.g., metaloporhirins), blockage of HO-1 could be a novel strategy for mobilizing HSPCs. Our recent in vivo mobilization studies lend support to this hypothesis. Disclosures: No relevant conflicts of interest to declare.

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C/EBPβ-Mediated Expansion of Hematopoietic Stem/Progenitors Precedes ‘Emergency’ Granulopoiesis Induced by Candidemia.

Blood ◽

10.1182/blood.v120.21.2336.2336 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2336-2336

Author(s):

Akihiro Tamura ◽

Hideyo Hirai ◽

Yoshihiro Hayashi ◽

Hisayuki Yao ◽

Satoshi Yoshioka ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Steady State ◽

Hematopoietic Stem Cells ◽

Post Infection ◽

Conflicts Of Interest ◽

Mrna Level ◽

Cathepsin G ◽

Hematopoietic Stem ◽

Myeloid Progenitors

Abstract Abstract 2336 Granulopoiesis, the process of granulocyte production in the bone marrow (BM), is tightly regulated to meet host demands during both 'steady state' and 'emergency' situations such as infections. The transcription factor CCAAT/Enhancer Binding Protein β (C/EBPβ) plays critical roles in emergency granulopoiesis (Hirai et al. Nat Immunology, 2006). However, the precise developmental stages in which C/EBPβ is required are unknown. In this study, we investigated the roles of C/EBPβ in the proliferation and differentiation of prospectively identified intermediates between hematopoietic stem cells and mature granulocytes in mouse BM. In order to analyze the mouse BM cells undergoing granulopoiesis, novel flow cytometric method was developed. Mouse BM cells retaining the ability to give rise to granulocytes were dissected into five distinct subpopulations (#1–#5) according to their levels of c-kit and Ly-6G expression. Upon infection of Candida albicans (4 × 106 CFU/20 g body weight/mouse) on day 1, C/EBPβ was upregulated at the protein level but not at mRNA level in all the granulopoietic subpopulations, suggesting the importance of the transcription factor in □emergency' granulopoiesis. Then, the role of C/EBPβ was further assessed by analyzing C/EBPβ knockout (KO) mice. At steady state, the distribution of granulopoietic cells in BM of C/EBPβ KO mice at □esteady state' was identical to that of wild type (WT) mice. In contrast, the rapid increase in immature subpopulations #1 and #2 observed in WT mice at 1 day post-infection was significantly attenuated in C/EBPβ KO mice. The levels of mRNA expression for granule proteins (cathepsin G, myeloperoxidase, elastase 2, proteinase 3, lactoferrin and MMP9) within each subpopulation from WT and C/EBPβ KO mice were identical at both steady state and during infection. When the cell cycle status of these models was evaluated using in vivo BrdU labeling experiments, incorporation of BrdU in subpopulation #1 and #2 in C/EBPβ KO mice was always slightly lower than in WT mice, but the differences were not statistically significant. These findings suggest that C/EBPβ is required for efficient proliferation of early granulocytic precursors but not directly involved in the differentiation/maturation process. To elucidate the roles of C/EBPβ in the proliferation of the early granulopoietic subpopulations, the hematopoietic stem cells (HSCs) and myeloid progenitor compartments were analyzed in WT and C/EBPβ KO mice. The frequency and number of c-kit+ Sca-1+ lineage markers− HSC were identical between WT mice and C/EBPβ KO mice during the steady state, and were not significantly affected on day 1 post-infection. Induction of candidemia increased the frequency and number of granulocyte-macrophage progenitors (GMP) in WT mice, and these increases were significantly attenuated in C/EBPβ KO mice. Upon induction of candidemia, the frequency of BrdU-positive cells in the HSC and common myeloid progenitors (CMP) populations from WT mice increased significantly; however, an increase of BrdU-positive cells was observed only within the HSC compartment in C/EBPβ KO mice, and at a lower level than that in WT mice Taken together, these data suggest that the proliferation of early granulocytic precursors is tightly coupled to differentiation/maturation and that C/EBPβ is involved in the efficient amplification of early granulocyte precursors including HSC and myeloid progenitors during candidemia-induced 'emergency' granulopoiesis. Disclosures: No relevant conflicts of interest to declare.

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The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils

Biochemical Journal ◽

10.1042/bj3110667 ◽

1995 ◽

Vol 311 (2) ◽

pp. 667-674 ◽

Cited By ~ 62

Author(s):

C Dahlgren ◽

S R Carlsson ◽

A Karlsson ◽

H Lundqvist ◽

C Sjölin

Keyword(s):

Membrane Fraction ◽

Plasma Membranes ◽

Subcellular Fractionation ◽

Secretory Vesicle ◽

Human Neutrophils ◽

Neutrophil Granulocytes ◽

Secretory Vesicles ◽

Membrane Glycoproteins ◽

Specific Granules ◽

Protein Group

The subcellular localization of two members of a highly glycosylated protein group present in lysosomal membranes in most cells, the lysosome-associated membrane proteins 1 and 2 (Lamp-1 and Lamp-2), was examined in human neutrophil granulocytes. Antibodies that were raised against purified Lamp-1 adn Lamp-2 gave a distinct granular staining of the cytoplasm upon immunostaining of neutrophils. Subcellular fractionation was used to separate the azurophil and specific granules from a light-membrane fraction containing plasma membranes and secretory vesicles, and Western blotting was used to determine the presence of the Lamps in these fractions. The results show that Lamp-1 and Lamp-2 are present in the specific-granule-enriched fraction and in the light-membrane fraction, but not in the azurophil granules. Separation of secretory vesicles from plasma membranes disclosed that the light-membrane Lamps were present primarily in the secretory-vesicle-enriched fraction. During phagocytosis both Lamp-1 and Lamp-2 became markedly concentrated around the ingested particle and they both appear on the cell surface when the secretory organelles are mobilized.

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Olfactomedin 4, An Indicator of the Existence of Neutrophil Subsets.

Blood ◽

10.1182/blood.v116.21.3775.3775 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3775-3775

Author(s):

Stine Novrup Clemmensen ◽

Sara Roervig ◽

Jonathan Wren ◽

Martin Illemann ◽

Andreas Glenthoj ◽

...

Keyword(s):

Bone Marrow ◽

Mrna Expression ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Subcellular Fractionation ◽

Human Neutrophils ◽

Specific Granules ◽

Blood Neutrophils

Abstract Abstract 3775 Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not. Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist. Disclosures: No relevant conflicts of interest to declare.

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The Expression of the Co-Inhibitory Molecule PD-1 and Its Ligand PD-L1 in Patients with MDS

Blood ◽

10.1182/blood.v126.23.5212.5212 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5212-5212

Author(s):

Suxia Geng ◽

Jianyu Weng ◽

Xin Huang ◽

Ping Wu ◽

Chengxin Deng ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Conflicts Of Interest ◽

Mrna Level ◽

Hematologic Malignancies ◽

Expression Level ◽

Activated T Cells ◽

Expression Levels ◽

Significant Difference ◽

Normal Controls

Abstract Programmed death receptor 1 (PD-1) is an important immunosuppressive molecule and expresses on activated T cells, B cells and myeloid cells. PD-L1, a primary ligand PD-1, is mainly expressed on T cells and primary B cell surfaces and plays a role in the differentiation and apoptosis of these cells, induces a coinhibitory signal in activated T cells, promotes T cells apoptosis, incompetence and functional exhaustion. The expression of PD-1 is high in patients with hematologic malignancies and a high expression of PD-L1 is found on hematologic malignancies cells. To further know the characteristics of PD-1 and PD-L1 in patients with MDS, we detected theexpression of PD-1 and PD-L1 in peripheral blood (PB) and bone marrow (BM) samples from 25 RAEB and 10 RARS patients using Real-time PCR. The PD1 and PD-L1 expression levels of 13 PB and 8 BM samples from normal individuals were as controls. The PD-1 levels of PB samples from 25 RAEB patients [42.40(4.5-173.96)%] were significantly higher than that from normal controls [32.32(19.45-41.38)%, P=0.026]. While the level of PD-1 in 10 RARS patients was comparable to that of normal controls and RAEB patients (P=0.401 and P=0.352). Compared to normal controls [23.72(3.23-39.2)%], the median PD-1 level of BM from 10 RAEB patients[36.81(12.14-151.52)%] showed an increasing tendency, but the difference was not statistically significant (P=0.062). PD-L1 expression levels of PB samples from RARS patients were significantly lower than that of normal controls (P=0.009). There were no significant difference between RAEB patients and normal controls about the PD-L1 expression level in PB and BM samples (P=0.248 and P=0.181) and between RAEB and RARS patients about PD-L1 expression level in PB samples (P=0.243). PD-1 level in BM samples from 11 RAEB patients with remission was (31.32±15.75)% and it was lower significantly than that before treatment [(59.94±47.44)%, P=0.034]. After progression or transformation, the expression level of PD1 (54.72±37.27)% increased again and was higher than that in remission (P=0.028).There were also no significant difference on PD-L1 expression among before treatment, bone marrow remission and progression or transformation (P>0.05). In 8 RAEB patients transformed to AML after treatment, PD-1 level has a decreasing tendency(P =0.05)and the change of PD-L1 has no significant difference (P>0.05). In conclusion, PD1 mRNA level increased significantly in patients with RAEB and the changes of PD-1 was associated with the evolution of the disease after treatment with demethylating agents. The level of PD1 may be used as an indicator to determine the efficacy, but the changes of PD-L1 was not found regularity. Disclosures No relevant conflicts of interest to declare.

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THE LOCALIZATION OF LIPIDS IN HUMAN BLOOD AND BONE MARROW CELLS

Blood ◽

10.1182/blood.v5.1.79.79 ◽

1950 ◽

Vol 5 (1) ◽

pp. 79-88 ◽

Cited By ~ 28

Author(s):

MARVIN L. BLOOM ◽

GEORGE B. WISLOCKI

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Ground Substance ◽

Metabolic Factors ◽

Baker’S Method ◽

Sudan Black B ◽

Specific Granules ◽

Neutrophilic Leukocytes ◽

Extraction Test ◽

Supravital Staining

Abstract By Baker’s method for the staining of lipids, various types of cytoplasmic rods and granules in leukocytes give a positive acid hematein test but are negative after the pyridine-extraction test. According to Baker,8 this combination of results indicates the presence of phospholipids. Some of the rods and granules which are stained appear to be mitochondria, judging from their shape, size and distribution. as well as from the fact that they coincide in all respects with mitochondria demonstrable by supravital staining. Baker8 pointed out that in a great variety of cells his method stained mitochondria, although he did not report having examined leukocytes. In addition to mitochondria, Baker’s method stains the specific granules of the three varieties of granular leukocytes. In the neutrophilic leukocytes, the granules appear gray or black; in some of the eosinophilic leukocytes, they are intensely black, while in others they are unstained although surrounded by stained ground substance; and in the basophilic leukocytes, some of the granules of individual cells are stained while others are not. Provided the method is chemically specific, it follows that the specific granules of these leukocytes contain phospholipids. The appearance of the eosinophils indicates that many of them possess granules composed of protein surrounded by phospholipid, although some of them contain granules with lipid in their interiors. These differences in the eosinophils suggest possible functional stages, related perhaps to the age of the cells or some other metabolic factors. In the monocytes, besides a general stippling of the cytoplasm corresponding to mitochondria, the region of the attraction sphere often contains numerous deeply stained granules. Sudan black B stains both the mitochondria and the specific leukocytic granules in a manner similar to Baker’s method. However, certain slight differences were noticed. Platelets were brought out moderately distinctly by the acid hematein procedure, whereas in sudan black preparations they were barely visible. A variability was noted in the staining of the granules of the eosinophilic leukocytes in Baker’s method, in contrast to the uniformity of their staining with sudan black. This difference should perhaps be interpreted as indicating that the eosinophilic granules contain other lipid substances besides phospholipids. Mitochondnia, in whatsoever type of leukocyte they are observed, appear to stain more deeply and distinctly by Baker’s method than with sudan black, a circumstance suggesting that mitochondria are very rich in phospholipids.

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Congenital neutropenia: neutrophil proliferation with abnormal maturation

Blood ◽

10.1182/blood.v46.5.723.723 ◽

1975 ◽

Vol 46 (5) ◽

pp. 723-734 ◽

Cited By ~ 41

Author(s):

RT Parmley ◽

M Ogawa ◽

CP Jr Darby ◽

SS Spicer

Keyword(s):

Bone Marrow ◽

Acid Phosphatase ◽

Colony Stimulating Factor ◽

Peripheral Blood Leukocytes ◽

Congenital Neutropenia ◽

Blood Leukocytes ◽

Specific Granules ◽

Primary Granules ◽

Stimulating Factor ◽

Myelin Figures

Abstract A child with congenital neutropenia was studied using bone marrow culture and ultrastructural and cytochemical techniques. The patient's marrow cells formed a large number of granulocytic colonies of normal size in culture, and her peripheral blood leukocytes produced adequate colony-stimulating factor. No serum inhibitors were identified. The patient's promyelocytes from direct marrow and culture appeared normal in ultrastructure, and primary granules, contained peroxidase and acid phosphatase activity. Myelocytes and rare segmented neutrophils from direct marrow specimens demonstrated atypical notched nuclei, myelin figures in Golgi lamellae and primary (azurophilic) granules, and no identifiable secondary (specific) granules. These data indicate an intrinsic neutrophil defect which allows normal proliferation of precursor cells, but results in abnormal granulogenesis and apparent inability to form secondary granules.

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miRNA-126 Inhibits Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells (BMSCs)

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2022.2943 ◽

2022 ◽

Vol 12 (4) ◽

pp. 794-799

Author(s):

Le Chang ◽

Wei Duan ◽

Chuang Wang ◽

Jian Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Mrna Level ◽

Alizarin Red ◽

Runx2 Expression ◽

Alp Activity ◽

Smad4 Protein ◽

Marrow Mesenchymal Stem Cells

This study was to determine whether microRNA (miRNA)-126 regulates osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were extracted and stimulated for osteogenic differentiation. Functional experiments were conducted to assess miR-126’s impact on BMSCs differentiation. Western blot and RT-qPCR determined miR-126 expression. ALP activity detection and alizarin red staining detection were also performed. After osteogenic differentiation of BMSCs, miR-126 expression was gradually decreased over time. Overexpression of miR-26 decreased ALP activity, Notch signaling activity as well as declined Runx2 expression and calcium Salt nodules after treatment. Importantly, we found that Smad4 serves as a target of miR-126 while upregulation of the miRNA was accompanied with the decreased Smad4 protein expression without affecting the Smad4 mRNA level. In conclusion, miR-126 restrains osteogenic differentiation through inhibition of SMAD4 signaling, providing a novel insight into the mechanism.

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