bluuebunny/crossref_metadata_2025 huggingface dataset:
Gfi1 Protein Turnover Is Regulated by the Ubiquitin Ligase Triad1.
Authors: van der Meer Laurens T. , Marteijn Jurgen A.F. , de Witte Theo M. , Jansen Joop H. , van der Reijden Bert A.
Published: April 2020

The transcriptional repressor Growth factor independence-1 (Gfi1) plays an essential role during various stages of hematopoiesis. It is crucial for the self-renewal and long-term reconstituting potential of stem cells, essential for neutrophilic differentiation, and it plays an important role in T-cell and dendritic cell development. Gfi1 has also been implicated in malignant hematopoeisis because the Gfi1 gene is a common proviral integration site in murine leukemia models. We recently found that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Although Gfi1 mRNA levels are low in primary human monocytes, the protein levels are high due to low proteasomal degradation. Conversely, in mature granulocytes Gfi1 mRNA levels are high but protein levels are low due to strong proteasome-mediated turnover. Because Gfi1 plays an important role in normal and malignant hematopoiesis it will be of great interest to identify the ubiquitin ligases that regulate its turnover.

Previously, we showed that the RING finger ubiquitin ligase Triad1 regulates myeloid cell proliferation. Using yeast-two-hybrid assays we found that Triad1 binds the zinc finger region of Gfi1. This interaction was confirmed in co-immunoprecipitation experiments. To study whether the turnover of Gfi1 is regulated by Triad1 we performed ubiquitination assays. To our suprise we found that instead of promoting ubiquitination, Triad1 inhibited Gfi1 protein ubiquitination, also in the presence of proteasome inhibitors. RNAi mediated down regulation of Triad1 protein levels stimulated Gfi1 ubiquitination. Importantly, expression of a Triad1 point mutant (H158A) that fails to bind the ubiquitin conjugating enzyme UbcH7 also inhibited Gfi1 ubiquitination. To study whether the observed diminished ubiquitination by Triad1 affected the turnover of Gfi1 we analyzed Gfi1 protein half-life using the protein synthesis inhibitor cycloheximide. This showed that Triad1 co-expression prolonged the half-life of Gfi1 significantly. We conclude that Triad1 inhibits Gfi1 ubiquitination, resulting in decreased turnover of the protein. As this inhibition also occurs in the presence of proteasome inhibitors and is independent of the ubiquitin ligase activity of Triad1, these data support a model in which Triad1 competes for Gfi1 binding with other ubiquitin ligases that do mark Gfi1 for proteasomal degradation. Currently, we are testing candidate ubiquitin ligases (RING finger and HECT proteins) that were found to associate with Gfi1 in yeast-two-hybrid assays to gain more insight in how the activity of this important transcription factor is regulated.
C/EBPα Binds and Activates the Distal PU.1 Enhancer.
Authors: Yeamans Christine , Wang Dehua , Paz-Priel Ido , Tenen Daniel G. , Friedman Alan D.
Published: April 2020

C/EBPα and PU.1 are both key regulators of myeloid development. C/EBPα (−/−) mice have markedly reduced granulocyte-monocyte progenitors, and PU.1 (−/−) mice lack B cells and monocytes and have greatly reduced neutrophil numbers. Genetic evidence indicates that higher levels of PU.1 are required for monocyte as compared to granulocyte development. Exogenous PU.1 or C/EBPα converts B-lineage to monocytic cells and increases monocyte as compared to granulocyte production from transduced myeloid progenitors cultured in IL3/IL6/SCF or in GM-CSF. We previously demonstrated that activation of C/EBPα-ER with estradiol induces PU.1 mRNA within 4 hr even in the presence of cycloheximide and that C/EBPα binds and activates the PU.1 promoter via a site conserved in the murine and human genes. However, binding to the promoter site in a gel shift assay was weak compared to the strong binding site in the neutrophil elastase promoter. To obtain further evidence for regulation of the PU.1 gene by C/EBPα, we have now investigated regulation of the 240 bp proximal segment of the PU.1 distal enhancer located at −14 kb. We focused on the proximal segment of the enhancer due to is strong transcriptional activating activity when linked to the PU.1 promoter, compared to the repressive effect of the distal enhancer segment. Five sites within the proximal enhancer (C1–C5) fit the C/EBP consensus site T(T/G)NNGNAA(T/G) or differed from this consensus by 1 bp. Each was evaluated for binding to C/EBPα expressed transiently in 293T cells. Site C1 bound C/EBPα weakly, and C2 and C5 each bound C/EBPα strongly. Binding of C/EBPα to the endogenous proximal enhancer segment was also demonstrated in the HF1 myeloid cell line using the chromosomal immunoprecipitation (ChIP) assay. HF1 cells were employed for this assay due to their high-level expression of C/EBPα. Sites C1, C2, and C5 were mutated individually within the enhancer, which was then linked to the PU.1 promoter and the luciferase cDNA. Mutation of C1 minimally reduced the activity of this reporter, mutation of C2 reduced activity 2-fold, and mutation of C5 reduced activity 5-fold, on average, upon transient transfection into the more readily tranfectable 32DPKCδ cell line. These data provide further support for the conclusion that C/EBPα directly activates the PU.1 gene and that induction of PU.1 by C/EBPα, potentially in cooperation with additional factors such as c-Jun, helps direct bipotent myeloid progenitors to commit to the monocytic lineage.
MicroRNAs 155, -221 and -222 Control Megakaryopoiesis at Progenitor and Precursor Level through Ets-1 Multitargeting.
Authors: Marziali Giovanna , Lulli Valentina , Coppola Simona , Romania Paolo , Fontana Laura , Liuzzi Francesca , Biffoni Mauro , Felli Nadia , Croce Carlo Maria , Pelosi Elvira , Peschle Cesare
Published: April 2020

It is generally accepted that microRNAs (miRs) control basic biological functions, such as cell proliferation and differentiation. However, only few targets of the ~ 300 known mammalian miRs have been validated so far, thus hampering delineation of miR-based control circuitries. Particularly, little is known on miR function in mammalian hematopoiesis. We have investigated by microarray and Northern blot evalutation miR expression profiles in human megakarypoiesis, as evaluated in cord blood hematopoietic progenitor cell (HPC) unilineage culture through the megakaryopoietic (MK) differentiation-maturation pathway. These studies indicated that miR-155, -221 and -222 are abundant in HPCs, but sharply decline during megakaryopoiesis. The decline may favour megakaryopoiesis by unblocking translation of key functional target protein(s). Bioinformatic analysis predicted that miR-155, -221 and -222 target Ets-1, a transcription factor up-regulated in Mk differentiation, which transactivates relevant Mk genes (e.g., TPO receptor, PF4, CD42 and von Willebrand factor). Consistently, in megakaryocytic cells the increase of Ets-1 protein expression coincides with the miR-155, -221 and -222 decrease. To find out whether Ets-1 mRNA is a possible target of miR-155, -221 and -222, we cloned segments of the 3′UTR of the Ets-1 gene downstream of a firefly luciferase ORF. Luciferase assay confirmed a direct interaction between each of these miRs and the 3′UTR of Ets-1. Functional studies showed that enhanced expression of these three miRs impairs proliferation, differentiation and maturation of MK cells, at least in part via enhanced degradation of Ets1 mRNA and down-modulation of Ets-1 protein. Similar results were obtained by RNA interference against Ets-1. Finally, HPCs transfected with miR-155, -221, and -222 showed a significant reduction of their Mk clonogenic capacity, suggesting that down-modulation of these miRs favours MK progenitor recruitment and commitment. Altogether, these studies indicate that a novel regulatory circuitry, based on miR-155, -221, -222 multitargeting Ets-1 mRNA leading in turn to transactivation of Mk-specific genes, plays a key role in the control of megakaryopoiesis at both progenitor and precursor level.
Identification of Common Molecular Pathways in Granulopoiesis Associated with C/EBP Epsilon and Gfi-1-Deficient Neutrophil Specific Granule Deficiency (SGD).
Authors: Khanna-Gupta Arati , Halene Stephanie , Sun Hong , Dahl Richard , Boxer Laurence A. , Berliner Nancy
Published: April 2020

Neutrophil specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections of the skin and respiratory system. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and exhibit defects in chemotaxis and bactericidal activity. A mouse model deficient for the transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) manifests a similar phenotype to SGD patients, and functional mutations in the C/EBPε gene have been identified in two patients with SGD. However, other patients with a similar disease phenotype do not have functional C/EBPε mutations, suggesting that other genetic abnormalities in myelopoiesis can lead to SGD. Studies in our laboratory on one such patient lacking a functional C/EBPε mutation demonstrated elevated protein levels of C/EBPε and significantly decreased levels of the transcription factor Gfi-1 (Growth factor independent-1) in peripheral blood neutrophils from this SGD patient. However, no mutation has been found thus far in the coding region of the Gfi-1 gene from the SGD patient. SGD can therefore be classified as C/EBPε negative or C/EBPε positive. We hypothesize that during granulopoiesis defects in a common molecular pathway occur in both C/EBPε- positive and -negative SGD resulting in the SGD phenotype. In order to gain insight into the molecular features of the two SGD phenotypes in the face of severe limitations on primary material from SGD patients, we have generated and characterized two cell line models. Using bone marrow from both C/EBPε−/− and Gfi-1+/− mice and their corresponding wildtype (WT) littermates, we generated EML (erythroid, myeloid, lymphoid) cell lines by transducing bone marrow with a retroviral vector expressing the dominant negative RARα and selecting for immortalized cells. Upon induction of the knock out and WT EML cell lines with all-trans retinoic acid (ATRA) to the neutrophil stage, we confirmed that both C/EBPε−/− and Gfi-1+/− cells share morphologic and functional abnormalities corresponding to their respective knockout mice, and hence to SGD. To further characterize the molecular defects in SGD, we have initiated microarray and proteomics analyses using RNA and protein prepared from both knockout cell lines. Using this approach, we may delineate defective pathways in both knock out cell types and correlate these observations to the specific SGD type. Our study should shed light on the relationship between Gfi-1 and C/EBPε during neutrophil development and should address the issue as to whether common or divergent molecular pathways are responsible for C/EBPε-negative and -positive SGD.
MicroRNA Function in Human Hematopoiesis: Identification of Lineage- and Stage-Specific Expression Profiles, Pivotal Targets and Regulatory Circuitries.
Authors: Chicarella Simona , Felli Nadia , Ferracin Manuela , Pelosi Elvira , Morsilli Ornella , Calin George Adrian , Liu Chang-Gong , Negrini Massimo , Croce Carlo Maria , Peschle Cesare
Published: April 2020

MicroRNAs (miRs) play a key role in the regulation of gene expression in normal and abnormal cell growth/differentiation, via suppression of translation or degradation of target mRNAs mainly by base pairing to their 3′ UTR sequences. However, little is known on miR expression/function in normal hematopoiesis. We have assayed the level of miRs in serum-free unilineage culture of cord blood (CB) CD34+ hematopoietic progenitor cells (HPC) through the erythroid (E), megakaryocytic (Mk), granulocytic (G) and monocytic (Mo) series at discrete stages of differentiation up to terminal maturation. The assay included control CB T cells. The analysis was performed by microarray and confirmed by Northern blot. Altogether, 49 miRs are expressed at significant levels in hematopoiesis. Cluster analysis indicates lineage-specific expression patterns. Furthermore, miR levels are mostly downmodulated through HPC differentiation/maturation. A group of 12 single or clustered miRs is sharply modulated (> 10-fold) in hematopoiesis according to specific patterns, thus suggesting their key functional role. This group includes:miR-223, downmodulated in the E series, but upmodulated in the other lineages;miR-221/222, miR-146 and miR-155 declining more sharply in E than other lineages;miR-17/20/106 and miR-25, miR-32, declining prevailingly in the G and Mo series;a miR group showing little downmodulation in the Mk or Mo lineages (miR-123, -130a or miR-181a, b, respectively);miR-99b, -100 downmodulated in all lineages;miR-210 upmodulated in all lineages.

Bioinformatic analysis suggests that these miRs have important targets, which have been in part verified by the luciferase target assay (see below). Relevant examples include miR-221/222 and -130a targeting kit receptor, miR-146 targeting SDFR/CXCR4, miR 181a, b, 99b and 100, 210 targeting diverse HOX genes (respectively, HOXB5, A1 and A3). Novel miR-based regulatory circuitries have been identified:(a) miR-221/222 modulate erythropoietic and erythroleukemia cell growth via kit receptor targeting (N. Felli et al, PNAS, 2005);(b) miR-17-5p/20a/106 function as a master gene complex controlling monocytopoiesis through AML1 targeting, leading to M-CSF receptor transactivation (L. Fontana et al, this Meeting);miR-155 and -221/222 control megakaryopoiesis at progenitor and precursor level through Ets-1 multitargeting (G. Marziali et al, this Meeting).
Elucidating the Oncogenic Potential of NPMc+ In Vitro and In Vivo.
Authors: Cheng Ke , Grisendi Silvia , Clohessy John G. , Majid Samia , Sportoletti Paolo , Pandolfi Pier Paolo
Published: April 2020

Nucleophosmin (NPM) is a nucleolar phosphoprotein that plays a key role in ribosome biogenesis, control of genomic stability and stabilization of tumor suppressors, such as ARF (Grisendi et al. Nature Review Cancer, 2006). We have recently shown that NPM can act as a tumor suppressor through the generation and characterization of an NPM hypomorphic series in vivo (Grisendi et al. Nature, 2005). NPM is one of the most frequent targets of genetic alterations in hematopoietic tumors, as about 60% of all adult Acute Myelogenous Leukemia with a normal karyotype harbor C-terminal frameshift mutations (NPMc+). As a consequence, NPMc+ aberrantly localizes to the cytoplasm of leukemic blasts.

It has been shown that NPMc+ can act as a dominant negative NPM mutant sequestering endogenous NPM to the cytosol. However, to date this mutant has never been shown to behave as an oncogene, which casts doubt on its role in leukemogenesis. To assess the oncogenic potential of NPMc+, we carried out soft agar and low-density seeding assays using primary mouse embryonic fibroblasts (MEFs) expressing NPMc+ in combination with a battery of oncogenes. Interestingly, we found that NPMc+, but not wild type NPM, cooperate with adenovirus E1A to transform primary MEFs in soft agar. NPMc+/E1A also consistently showed high efficiency at forming foci in low-density seeding assays comparable to RasV12/E1A. However, NPMc+ does not transform alone or in combination with RasV12 or c-Myc and does not transform p53−/−, p21−/− or Arf−/− primary MEFs. This suggests that two complementary growth signals from NPMc+ and E1A can cooperate to evade cellular senescence and apoptosis. These data are consistent with a model by which NPMc+, through the inhibition of NPM function, can block the ARF response elicited by E1A. In support of these observations, we found that in primary cells from our NPM hypomorhic series Arf level and localization are dramatically affected by the progressive reduction in Npm dose. To elucidate in vivo the crosstalk between NPM and ARF, genetic crosses using Npm+/− and Arf+/− mice are currently ongoing and data from this analysis will also be presented.
Constitutive Activation of Hemostasis in Children with Sickle Cell Disease.
Authors: Steele MacGregor , Williams Suzan , Pluthero Fred , Bang K.W. Annie , Goldman Ran , Friedman Jeremy , Freedman John , Kirby Melanie , Rand Margaret L. , Blanchette Victor S. , Kahr Walter H.
Published: April 2020

Recent studies of sickle cell disease (SCD) suggest that hemostatic activation is a key aspect of pathophysiology, leading to clinical consequences such as vaso-occlusive crises and stroke. In many of these studies a SCD-associated hypercoagulable state has been inferred from markers suggestive of increased hemostatic activity, such as elevated levels of plasma thrombin-antithrombin complexes and tissue factor-containing microparticles. As part of a study using a broad-spectrum approach to explore the relationships among various aspects of normal and abnormal hemostasis in SCD we used a whole-blood coagulation assay (thromboelastography, TEG) to directly assess global hemostatic activation in children with SCD defined by genotype (SS and SC), together with a group of unaffected children. We also assessed platelet activation and procoagulant surface expression in platelets and red blood cells (RBCs) using flow cytometry. Eligible SCD subjects included:patients with painful crisis assessed at two time points: hospital admission (crisis group) and clinic follow-up appointment (follow-up group);patients not in crisis attending a regular clinic appointment (steady-state group).

Patients were ineligible if they had received a recent blood transfusion, hydroxyurea, anticoagulants or aspirin. The results of TEG assays with citrated whole blood showed that compared to SC patients (n = 16) and normal children (n = 16), SS patients (n = 45) had significantly (p<0.001) earlier clotting onset (mean R times were 4.5, 6.5 and 7.6 minutes for SS, SC and normals respectively) and significantly (p<0.001) higher rates of clotting (mean maximum clotting rates were 16.8, 12.6 and 10.9 mm/min for SS, SC and normals respectively). TEG clotting onset and maximum clotting rates were not significantly different among steady-state, crisis and follow-up groups of children with SCD (both SS and SC genotypes), nor between sexes within each study group. Whole blood flow cytometry revealed that platelet GPIIb/IIIa activation (PAC-1 binding) was significantly elevated (p<0.05) in SCD patients relative to normal children. In addition, markers of RBC procoagulant surface expression (RBC annexin A5 binding) and RBC-platelet aggregates were elevated in SCD patients compared to normal children. These results indicate that children with the SS genotype have an activated hemostatic system relative to normal and SC genotype children, and that this hypercoagulable state is maintained during sickle cell crisis as well as during steady state. It remains to be determined whether pharmacological interventions and/or RBC transfusions which improve clinical outcomes in SCD patients modify their constitutively hypercoagulable state.
Gene Expression Profiles during the γ- to β-Globin Switch in Erythroid Progenitors.
Authors: Li Wei , Pace Betty S.
Published: April 2020

The design and evaluation of therapies for sickle cell disease rely on our understanding of the sequential expression of the β-like globin genes during development. Drug inducers that reactivate γ-globin transcription after birth ameliorate symptoms in sickle cell patients. Therefore, the objective of our study is to identify trans-factors involved in γ-gene activation during the γ- to β-globin switch recapitulated in erythropoiesis. We previously established the one-phase liquid culture system using buffy coat mononuclear cells grown in erythropoietin (Epo; 4U/mL), stem cell factor (50ng/ml) and Interleukin-3 (IL-3; 10ng/ml). We observed high γ-globin transcription on day 7, equal γ- and β-globin mRNA levels by day 20 followed by high β-globin expression on day 28. Cell surface markers were measured by quantitative PCR (qPCR) and correlated with globin gene expression. During lineage commitment and erythroid maturation CD34+ cells decreased while the erythroid markers CD71, glycophorin A and Epo receptor increased 12 to 144-fold, followed by a decrease on day 28. We concluded that the γ- to β-globin switch was recapitulated in our system. Subsequent studies were performed with microarray and gene profiling by principle component analysis (PCA) to characterize proteins involved in switching. The Discover Chip from ArrayIt (Sunnyvale, CA) containing 380 human genes was used. cDNA from day 0/7, 7/21 and 21/28 were labeled with Cy3 and Cy5 respectively and used for standard chip hybridization. Fluorescence data was acquired using the Axon scanner and GenePixPro 6.0 software. By day 7, 88 genes were silenced and 68 activated; at day 28 an additional 38 genes were silenced and 47 activated. PCA defined the four major gene profiles consistent with developmental globin gene regulation. Profile 1 genes including IL-3, IL-9, heat shock protein 1a and neurofibromin were activated greater than 2-fold on day 7 suggesting positive regulators of γ-globin; trans-activators were not identified. Profile 2 genes were highly expressed around the switch on day 20. GATA-1, guanine nucleotide binding protein, ZF protein 132 and ERK1 expression peaked during this time. This data is consistent with a γ-globin silencer role previously demonstrated for ERK. Profile 3 genes also peaked on day 20 but continued to be highly expressed during β-globin transcription; EKLF, IL-8, Ras, Fos, and histone H3 were identified in this group. Experimental data support EKLF binding in the locus control region to compete with γ-globin expression. Finally Profile 4 genes increased steadily and peaked on day 28 consistent with positive β-globin regulators including NF-E2, ATF-2, IL-12, and thymosin β10. The microarray data were confirmed by qPCR in 90% of the genes tested. Our findings support the one-phase erythroid liquid culture system as a model for globin switching. We will expand gene profiling efforts using the Agilent 44K chip and PCA to confirm this model.
A Monoclonal Antibody to Lactadherin Inhibits Sickle Red Blood Cell Adhesion to Vascular Endothelial Cells in a Plasma Milieu.
Authors: Dasgupta Swapan K. , Guchhait Prasenjit , Le Anhquyen , Yellapragada Sarvari , Lopez Jose , Thiagarajan Perumal
Published: April 2020

Adherence of red blood cells to the endothelium initiates vaso-occlusion in sickle cell anemia. The increased adhesiveness of sickle erythrocytes is accompanied by several changes in the lipids of the erythrocyte membrane, including increased expression of phosphatidylserine (PS). One important PS-binding protein is lactadherin (also known as milk fat globule-EGF factor 8), a 45-kDa glycoprotein containing an Arg-Gly-Asp (RGD) sequence. It is secreted by macrophages and is present in normal plasma. Lactadherin promotes phagocytosis of PS-expressing apoptotic lymphocytes and sickle red blood cells by anchoring them to integrins on macrophages. Here, we investigated the role of endogenous lactadherin in adhesion of sickle erythrocytes to the endothelium. We developed a murine monoclonal antibody to human lactadherin, called L688, and investigated its effect on the adhesion of sickle red blood cells to histamine-stimulated human umbilical vein endothelial cells under hydrodynamic flow. In three experiments using washed erythrocytes resuspended in autologous plasma from three different patients with sickle cell anemia, L688 (20 μg/ml) inhibited adhesion by 24–30% (p<0.01). Further evidence for an important role for lactadherin in sickle erythrocyte adhesion to endothelial cells was provided by the observation that exogenous lactadherin enhanced adhesion in a concentration-dependent manner. Lactadherin-mediated adhesion was also inhibited by monoclonal antibody abciximab, (c7E3, 10 μg/ml) which targets the β3 integrin subunit common to both αIIbβ3 and αVβ3. Control antibodies had no effect. Finally, the lactadherin-dependent adhesion of sickle erythrocytes to activated endothelium was inhibited by PS vesicles but not by phosphatidylcholine vesicles, confirming an important role for PS in sickle cell adhesion. Consistent with this, normal erythrocytes can be induced to adhere to stimulated HUVEC in a lactadherin-dependent manner by treatment with N-ethylmaleimide (10 mM) and calcium ionophore A23187 (4 μM) — treatment that exposes PS on the outer leaflet of the red cell membrane. Together, these results indicate that lactadherin mediates sickle cell adhesion to the endothelium by bridging PS on the erythrocytes with αVβ3 integrin on the endothelium. We propose that anemia in sickle cell disease is at least partially due to phagocytosis of lactadherin-coated sickle erythrocytes in the spleen, liver, and lymph nodes. Those erythrocytes that are not ingested immediately by macrophages will become more adhesive for endothelium. Thus, lactadherin appears to be involved both in sickle cell clearance from the circulation and in adhesion to endothelium.
Activation of NK Cells Induces CD4 Expression and Allows HIV−1 Infection.
Authors: Harada Hideki , Goto Yumi , Dessouki Omar F. , Suzu Shinya , Okada Seiji
Published: April 2020

Natural killer (NK) cells play critical roles in immune surveillance without deliberate prior sensitization and restriction by major histocompatibility complex (MHC). Although function and cell number of NK cells are influenced in AIDS patients, direct interaction between HIV and NK cells is still controversial. Because steady-state NK cells are negative for CD4 which is a key molecule for HIV infection. In this study, we established a condition inducing CD4 expression and HIV-1 infection of NK cells in vitro.

CD4 was expressed on NK cells co-cultured with HFWT cells (NK cell-selective stimulating feeder cells) and IL-2. There were no differences in expression level of NK receptors, adhesion molecules and cytotoxicity between CD4+ and CD4- NK cell subpopulations. However, expression of activation markers, CD25 and HLA-DR, and size/granularity of the CD4+ NK cells were higher than CD4- NK cells. CD4+ NK cells expressed co-receptors for HIV-1, CCR5 and CXCR4. Thus, CD4 is induced on NK cells by activation, and the CD4+ NK cells are the possible target for HIV.

Next, we exposed HIV-1 clone (JR-FL) to the HFWT-activated NK cells, however, direct HIV infection to the NK cells was not observed. While, co-culture of activated NK cells with HIV infected T cells allowed HIV infection of NK cells. Because NK cell-specific marker, NKp46, was detected on p24+CD3-CD56+ cells but not on CD3+ cells, p24+CD3-CD56+ cells were certainly NK cells. These results demonstrate that NK cells are the targets of HIV.

Altogether, our data suggest that CD4+ T cells transfer HIV to NK cells during this cell-to-cell contact, which cause the NK cells to be the long-living viral reservoirs or modify the function of NK cells in HIV-infected patients. Our novel co-culture system using activated NK cells and HIV-infected T cells is the powerful tool to study the function of NK cells on HIV infection.
Novel Function of the TAL1/SCL Transcription Factor in Differentiation of Murine Bone Marrow Monocytes.
Authors: Dey Soumyadeep , Shi Yubin , Brandt Stephen J.
Published: April 2020

The TAL1 (or SCL) gene encodes a basic helix-loop-helix transcription factor that can activate or repress gene expression, depending on its interaction partners. TAL1 proteins are expressed in the vascular and hematopoietic systems and in erythroid, megakaryocytic, and mast cell precursors. TAL1 is essential for hematopoietic commitment and vascular remodeling during embryogenesis and is also important to the differentiation of erythroid and megakaryocytic progenitors postnatally. Although a mouse Tal1 cDNA was cloned from a bone marrow (BM) macrophage cDNA library and we reported expression of Tal1 protein in mouse bone marrow mononuclear cells (Blood83:1200, 1994), no studies have examined the function of this transcription factor in the monocyte-macrophage lineage. To address this issue, we characterized Tal1 expression in primary mouse BM monocytes and M1 monocytic leukemia cells induced to differentiate in vitro. We isolated BM cells from wild-type C57BL6/J mice and purified a mononuclear cell population by pronase treatment and density gradient centrifugation on horse serum. Monocytes were then induced to differentiate in culture, initially in the presence of both interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF) and subsequently in the presence of L-cell conditioned medium. Tal1 expression was maintained in these cultures, declining by only 14% over the full culture period, while calcitonin receptor expression, a marker of osteoclastic differentiation, was extinguished. In parallel, interleukin-6 receptor (IL-6R) and macrophage colony-stimulating factor receptor (M-CSFR) mRNA increased 3.5- and 114-fold, respectively. In M1 cells induced to differentiate with recombinant murine IL-6, Tal1 protein abundance decreased similarly with differentiation. To assess the function of Tal1 in this cell lineage, a full-length Tal1 cDNA was introduced into both primary BM monocytes and M1 cells via retroviral gene transfer. Puurified monocytes were transduced 24 hr after isolation with wild-type Tal1 or a DNA-binding-defective Tal1 mutant, T192P, in the MSCV-GFP vector. GFP-expressing cells were then sorted and induced to differentiate. Gene expression analysis using semi-quantitative PCR carried out after 8 days of culture showed increased expression of Tal1 mRNA in cells transduced with the wild-type Tal1 cDNA compared to the vector control. This was accompanied by upregulation of IL-6R and M-CSFR expression relative to both parental vector- and mutant-transduced cells. Northern blot analysis of M1 cells transduced with the Tal1 cDNA showed similar upregulation of M-CSFR mRNA relative to vector control cells. In summary, these studies show that the TAL1 transcription factor is expressed by murine monocytes during differentiation to macrophages and suggest a role for TAL1 in both gene expression and differentiation of this lineage. Further, they suggest a requirement for direct TAL1 DNA binding.
Adiponectin Produced by Lymphocytes Inhibits Granulopoiesis.
Authors: Crawford Lisa J. , Peake Roy W. , Price Susan , Morris Treen C. , Irvine Alexandra E.
Published: April 2020

Previous studies by our group have shown that normal unstimulated lymphocytes produce a protein which inhibits colony formation of granulopoietic progenitors, but has no effect on erythroid progenitors. Therefore, this inhibitor was initially designated GIA (granulopoietic inhibitory activity). GIA was identified as a glycoprotein of approximately 30 kDa, with a pI of 7.9 - 8.4. Futhermore, we demonstrated that this inhibitor may have physiological significance in that its production is altered in patients with neutropenia. GIA has proved difficult to characterise to date since it is produced in relatively low amounts although it has a high specific biological activity. Adiponectin is an adipokine reported to share many of the inhibitory characteristics of GIA and has been demonstrated to act as a negative regulator of hematopoiesis and immune response. This study aimed to determine whether GIA is adiponectin or if it represents an adiponectin-like molecule. Lymphocyte conditioned medium (LCM) from lymhocytes cultured at 1 X 106 cells/ml in HL-1 minimal medium was used as a source of GIA. Inclusion of LCM as 10% of the top layer of agar in a myeloid colony assay inhibited growth of CFU-GM by 52 ± 11 % (n=3), confirming the presence of the inhibitory activity. RNA and protein from lymphocytes and LCM harvested over a 7 day culture period were subsequently investigated for adiponectin expression. Western blot analysis demonstrated a distinct banding pattern in days 3-7 LCM corresponding to monomers, dimers, trimers and greater. This is consistent with adiponectin which circulates as a multimer of trimers. Characterisation of GIA at the transcript level confirmed that GIA is in fact adiponectin. The N-terminal collagenous domain, C-terminal globular domain and full length adiponectin were amplified by RT-PCR analysis. Adiponectin is thought to be secreted exclusively from adipocytes and much of our current knowledge of this molecule relates to its metabolic functions. Our study provides evidence that adiponectin is also produced by lymphocytes and may play a role in the pathogenesis of neutropenia.
Abnormal Localization of Neutrophil Elastase in Patients with Severe Congenital Neutropenia.
Authors: Sato Takashi , Habara Masakazu , Kihara Hiroki , Kawaguchi Hiroshi , Miki Mizuka , Nakamura Kazuhiro , Okada Satoshi , Ishikawa Nobutsune , Ohno Norioki , Kobayashi Masao
Published: April 2020

Mutations in the ELA2 gene encoding neutrophil elastase (NE) in patients with severe congenital neutropenia (SCN) are involved in the pathogenesis of this disorder, possibly due to the abnormal protein trafficking and accelerated apoptosis of myeloid cells. In this study we precisely examined the localization of NE in neutrophils and myeloid precursor cells in bone marrow in patients with SCN using immunofluorescence microscopy equipped with three-dimensional analysis program. Three patients with SCN were enrolled in this study. All patients with SCN showed heterozygous mutation in the ELA2 gene. In normal subjects the pattern of localization of NE in mature neutrophils was almost similar to those of myeloperoxidase (MPO), proteinase 3, lysosomal associated membrane protein 2 (LAMP2). Administration of G-CSF to normal subjects did not affect the pattern of the localization of these proteins in neutrophils. In contrast, mature neutrophils elicited by the administration of G-CSF in patients with SCN NE predominantly localized to the plasma membranes. A small part of NE was detected in the cytoplasmic compartment. The pattern of localization of NE was significantly different from those of MPO, proteinase 3, and LAMP2 in SCN patients, suggesting the abnormal traffic of NE to granules. Adaptor proteins 3 (AP3) specifically shuttles transmembrane cargo proteins from the trans-Golgi to lysosomes. AP3 of myeloid progenitor cells enriched for CD33-positive cells in normal bone marrow was localized in both cytoplasm and plasma membranes. The localization pattern of AP3 was completely consistent with those of NE, MPO, and LAMP2. The localization of AP3 of promyelocytes in patients with SCN was observed in both plasma membranes and cytoplasm. This finding was completely similar to that in normal myeloid precursor cells. However, the localization of NE of promyelocytes in SCN patients was predominantly in plasma membrane. The figures merged apparently presented the different localization of NE and AP3. This result was confirmed by the 3-dimensional analysis with histogram. The localizations of other constituents of primary granules, MPO, poteinase 3, and LAMP2, were consistent with those of AP3. These observations suggest that the mislocalization of NE in myeloid precursor cells in SCN patients does not result from a generalized impairment of protein trafficking but is specific to the mutant NE. The abnormal localization of NE may be involved in the pathogenesis of SCN associated with ELA2 mutation.
Primary Treatment of Waldenstrom’s Macroglobulinemia (WM) with Dexamethasone, Rituximab and Cyclophosphamide.
Authors: Dimopoulos Meletios A. , Anagnostopoulos Athanasios , Kyrtsonis Marie Christine , Tsatalas Constantinos , Kokkini Garyfallia , Repoussis Panagiotis , Zomas Athanasios , Symeonidis Argiris , Michali Evridiki , Anagnostopoulos Nikolaos , Terpos Evangelos , Economopoulos Theophanis , Pangalis Gerasimos
Published: April 2020

Introduction: The three main choices for the primary treatment of WM are alkylating agents, nucleoside analogues and rituximab. There is evidence that combinations of nucleoside analogues and rituximab are highly effective. However, when such regimens are administered, there are concerns regarding myelosuppresion, immunosuppression and impact on stem cell collection. We designed a phase II study which included previously untreated WM patients (with clearly defined criteria for initiation of treatment) who were treated with a combination of dexamethasone, rituximab and cyclophosphamide (DRC).

Patients and methods: Patients with WM received primary treatment with dexamethasone 20mg i.v. followed by rituximab 375mg/m2 i.v. on day 1 and cyclophosphamide 100mg/m2 p.o. bid days 1–5 (total dose 1000mg/m2). This regimen was repeated every 21 days for 6 courses.

Results: Since July 2002, 70 patients have been enrolled in this multicenter study. As of July 2006, 60 patients have completed the scheduled courses of treatment. Patients characteristics include: median age of 71 years (range 33 to 89 years), 63% male, 28% B symptoms, 32% splenomegaly, 40% lymphadenopathy, 25% hyperviscosity syndrome, 62% hemoglobin <10g/dL, 43% serum monoclonal protein ≥4gr/dL, 36% serum albumin <3,5g/dL, 40% serum β2 microglobulin >4mg/dL. On an intent to treat basis, 70% of patients achieved at least 50% reduction of serum monoclonal protein concentration, including 7% of patients who achieved immunofixation-negative complete response. Furthermore in 20% of patients <50% reduction of monoclonal protein or disease stability was observed, whereas progressive disease during treatment was documented in only 10% of patients. The median time to response was 4 months (range 0,7 months to 14 months). With a median follow-up of 24 months, 60% of patients are progression-free. With the exception of one patient who died of interstitial pneumonia, the DRC regimen was well tolerated. 20% of patients developed grade 3–4 neutropenia. Severe opportunistic infections were not observed. Mild or moderate effects related to rituximab infusion such as fever, rigors and headache were seen in 20% of patients. In patients where stem cell collection was attempted, this procedure was successful in all

Conclusion: Our large, multicenter trial showed that the non-stem cell toxic DRC regimen is an active and well tolerated treatment for symptomatic patients with WM. With this treatment, disease control can occur in the majority of patients without the risks of myelosuppression and immunosuppression which may occur when nucleoside analogues are used. This regimen may be particularly useful when high-dose therapy is an option for patients with WM.
Prospective Isolation of Human Eosinophil-Committed Progenitors.
Authors: Mori Yasuo , Iwasaki Hiromi , Yoshimoto Goichi , Okeda Aki , Miyamoto Toshihiro , Mizuno Shin-ichi , Harada Mine , Takatsu Kiyoshi , Akashi Koichi
Published: April 2020

Eosinophils play an important role in the pathogenesis of allergic reactions or chronic inflammatory diseases by releasing various types of cytokines and chemical mediators. Recently, we have identified murine eosinophil-committed progenitors (mEoPs) in mouse bone marrow. The expression of receptor for IL-5, a critical cytokine for proliferation and differentiation of eosinophils, was a key marker to isolate mEoPs: mEoP was IL-5Ra+Lineage(lin)-CD34+c-Kitlow population in murine bone marrow (J Exp Med.201, 1891ndash;7, 2005). Here we report that EoPs are prospectively isolatable also in human bone marrow. We analyzed the expression of human IL-5Ra in human stem and progenitor populations, and found that a fraction of common myeloid progenitor (CMP; lin-CD34+CD38+CD45RA-IL-3Ra+) population expressed hIL-5Ra on their surface by using anti-human IL-5Ra monoclonal antibodies. IL-5Ra protein and mRNA were undetectable in hematopoietic stem cells (HSCs; lin-CD34+CD38-), common lymphoid progenitors (CLPs; lin-CD34+CD38+CD10+), megakaryocyte/erythrocyte progenitors (MEPs; lin-CD34+CD38+CD45RA-IL-3Ra-), or granulocyte/monocyte progenitors(GMPs; lin-CD34+CD38+CD45RA+IL-3Ra+) by FACS and RT-PCR, respectively. The IL-5Ra+ cells within the CMP fraction constituted only ~0.04% of steady-state bone marrow mononuclear cells, and gave rise only to pure eosinophil colonies. Thus we termed this population as human EoP (hEoP). Both HSCs and the IL-5Ra- fraction of CMPs gave rise to IL-5Ra+ hEoPs in vitro in the presence of IL-3 and GM-CSF, while MEPs or GMPs never generated hEoPs, indicating that human eosinophil pathway diverges at the CMP stage, and that the eosinophil potential was lost at the GMP or MEP stage. Accordingly, the human eosinophil pathway is different from that in murine hematopoiesis where mEoPs develop from the GMP stage. Strikingly, the number of hEoPs in the bone marrow of patients with hypereosinophilic syndrome was significantly (~4-fold) increased as compared to that in normal bone marrow, suggesting that hEoP represents a critical stage for eosinophilia in vivo. Thus, the hEoP is an attractive candidate for therapeutic target in eosinophil-related allergic and inflammatory disorders. This population might also be very useful to study the molecular mechanism of human eosinophil development.
Kruppel-Like Factor 4 Upregulates p21 and Downregulates Proliferation of Human and Mouse HSPCs, but Is Not Essential for Mouse HSPC Repopulation.
Authors: Alder Jonathan K. , Georgantas Robert W. , Hildreth Richard L. , Yu Xiaobing , Civin Curt I.
Published: April 2020

Several Kruppel-like factor family members, including KLF1, KLF2, KLF3, and KLF6 have pivotal roles in hematopoiesis. Experiments in zebrafish have suggested that KLF4 may play a similar role. Here we found that enforced expression of KLF4 in hematopoietic cells induced cell cycle arrest without triggering apoptosis. Based on the high levels of expression of KLF4 in mouse and human hematopoietic stem-progenitor cells (HSPCs), we hypothesized and demonstrated that KLF4 regulates proliferation of these cells through regulation of p21cip1/waf1 (p21). Nevertheless, KLF4−/− mouse fetal liver cells had normal numbers of all mature lineages and provided radioprotection, similar to wild type (wt) controls. Furthermore, in long-term competitive repopulation assays, KLF4−/− mouse HSPCs demonstrated hematopoietic potency equivalent to wt. We found that KLF2 is expressed at higher levels than KLF4 in mouse HSPCs and is a more potent activator of p21, suggesting that KLF2 (and/or other KLF family members) may play a compensatory role in KLF4−/− HSPCs. Thus, although is not essential for their normal development and function, KLF4 expression is sufficient to induce p21-mediated cell cycle arrest in hematopoietic cells.
Identification of Key Cellular Regulators That Interact with Id1 To Control Hematopoietic Stem Cell Behavior.
Authors: Jankovic Vladimir , Ciarrocchi Alessia , DeBlasio Tony , Benezra Robert , Nimer Stephen D.
Published: April 2020

The ability of hematopoietic stem cells to tightly regulate the transition from relative quiescence and self-renewal to the transiently amplifying, differentiating progenitor fate is critical for HSC homeostasis as well as their regenerative capacity. We have recently described the diminished frequency and rapid exhaustion of HSC self-renewal capacity in the absence of the dominant negative helix-loop-helix molecule Id1. Furthermore, Id1 null HSCs have an increased rate of cycling, coupled with accelerated myeloid commitment both in vivo and in vitro. This is reflected in the elevated expression of myelo-erythroid transcription factors (c/EBPalpha and GATA1) within the Lin−c-kit+Sca-1+ population - “myeloid priming”. The major targets of Id1 mediated transcriptional repression are the ubiquitous E protein E2A as well as Ets transcription factors (Ets1 and Ets2). We hypothesized that the unrestrained activity of these and/or other targets of Id1 transcriptional repression leads to premature HSC commitment in Id1 null animals. Indeed, we show that HSC differentiation in culture can be delayed by transduction of E2A directed shRNA specifically in Id1 null, but not in wild-type Id1 expressing cells. This indicates an abnormal E2A activity in Id1 null HSCs that could be responsible for their increased differentiation status. To further define the transcriptional deregulation in Id1 null HSCs, we have used the Affymetrix microarray technology. We observed ~3 fold increased expression of the CDK inhibitor p21 in freshly isolated Id1 null HSCs and have confirmed this result by multiple independent qPCR measurements. The transcriptional induction of p21 by E2A as well as its repression by Id1 have been well established. Therefore, the observed p21 induction could be explained by the elevated level of E2A activity in HSCs in the absence of Id1 expression. To explore the functional significance of Id1 mediated p21 regulation in HSCs, we have generated p21/Id1 double knockout animals. Surprisingly, despite its reported function in restricting the cell cycle entry of normal HSCs, we show that in the context of Id1 loss, p21 expression is required for the accelerated HSC cycling, and unlike Id1 single null HSCs, p21/Id1 double knockout HSCs do not show accelerated myeloid differentiation in culture. Therefore, we propose that Id1 actively represses E2A activity in HSCs, as well as the induction of p21, which could be an important component of the HSC commitment program. Further studies will be presented defining the in vivo relevance of the Id1/p21 genetic interaction for HSC growth and differentiation.
Dose Dependent Effects of STAT5 on Proliferation, Differentiation and Self Renewal of Hematopoietic Stem/Progenitor Cells.
Authors: Wierenga Albertus T.J. , Rozenveld-Geugien Marjan , Vellenga Edo , Schuringa Jan Jacob
Published: April 2020

To study the dose dependent effects of ectopically expressed STAT5A in cordblood (CB) CD34+ cells, we developed a tamoxifen-inducible STAT5A expression system. Constitutively active STAT5A (STAT5A1*6) was fused to the ER domain to facilitate its activation upon stimulation of the transduced cells by the addition of 4OHT. Transactivation and DNA binding studies revealed that STAT5A could be activated to different levels in a controlled way by addition of different concentrations of 4OHT. CB CD34+ cells were retrovirally transduced with this construct and the effects on proliferation, differentiation and self renewal was studied.

In an MS5 coculture system, we demonstrated that a high dose of 4OHT (500nM) resulted in a rapid burst of erythropoiesis and a block in myeloid differentiation (as previously demonstrated using STAT5A1*6 without an ER domain). Moderate levels of 4OHT (20nM) however, resulted in a much higher proliferation of not yet differentiated cells, ultimately resulting in a higher expansion. This higher expansion coincided with a higher frequency of early (day10) cobblestones (without 4OHT: 1,5 cobblestones per 104 plated cells, 20nM 4OHT: 1750/104, 500nM 4OHT:200/104). RT-PCR of STAT5A1*6-ER transduced cells revealed a rapid downregulation of C/EBPalpha mRNA upon stimulation with 4OHT, with a dose response curve inversely correlated to the number of early cobblestones, suggesting that these two processes are linked.

When STAT5A1*6-ER transduced cells were cultured in methylcellulose, stimulation with increasing concentrations of 4OHT resulted in decreasing numbers of colonies (myeloid as well as erythroid). Interestingly, whereas colonies from unstimulated (control) cultures and 500nM 4OHT stimulated cultures could not be secondary plated, cultures stimulated with only very low concentrations of 4OHT (1 nM) could be secondary plated, giving rise to only myeloid colony formation. These findings demonstrate that the level of activation of STAT5 affects the proliferation and differentiation in a separate manner; low levels of STAT5A activation results in self renewal of the progenitor/stem cell pool cells, whereas high levels of STAT5A induces a rapid differentiation at the expense of proliferation and self renewal.

These data correspond well to our findings in AML cells. Downregulation of the moderate constitutive STAT5 activation in CD34+ AML cells by STAT5 RNAi affects especially the proliferative capacity of the AML cells. In conclusion, we hypothesise that low constitutive levels of active STAT5 might contribute to leukemic transformation.