Late Precursors Research Articles

Stage-specific GATA3 induction promotes ILC2 development after lineage commitment

Group 2 innate lymphoid cells (ILC2s) are a subset of innate lymphocytes that produce type 2 cytokines, including IL-4, IL-5, and IL-13. GATA3 is a critical transcription factor for ILC2 development at multiple stages. However, when and how GATA3 is induced to the levels required for ILC2 development remains unclear. Herein, we identify ILC2-specific GATA3-related tandem super-enhancers (G3SE) that induce high GATA3 in ILC2-committed precursors. G3SE-deficient mice exhibit ILC2 deficiency in the bone marrow, lung, liver, and small intestine with minimal impact on other ILC lineages or Th2 cells. Single-cell RNA-sequencing and subsequent flow cytometry analysis show that GATA3 induction mechanism, which is required for entering the ILC2 stage, is lost in IL-17RB+PD-1− late ILC2-committed precursor stage in G3SE-deficient mice. Cnot6l, part of the CCR4-NOT deadenylase complex, is a possible GATA3 target during ILC2 development. Our findings implicate a stage-specific regulatory mechanism for GATA3 expression during ILC2 development.

PETROLEUM GEOLOGY OF THE CENOZOIC SUCCESSION IN THE ZAGROS OF SW IRAN: A SEQUENCE STRATIGRAPHIC APPROACH

The Cenozoic stratigraphy of the Zagros records the ongoing collision between the Arabian and Eurasian Plates and the closure of NeoTethys. A Paleogene NW‐SE trending foreland basin was inherited from a Late Cretaceous precursor. Widespread progradation into the foredeep was a feature of both margins which, allied to ongoing tectonism, had by the late Eocene led to the narrowing and subsequent division of the foredeep into the Lurestan – Khuzestan and Lengeh Troughs, separated by the northward continuation of the rejuvenated Qatar‐Fars Arch. This sub‐division strongly influenced subsequent deposition and the petroleum geology of the area. In addition, the diachronous nature of the Arabian – Eurasian collision led to strong diachroneity in lithostratigraphic units along the length of the Zagros. Hence its petroleum geology is best understood within a regional sequence stratigraphic framework. This study identifies three tectono‐megasequences (TMS 10, TMS 11a, TMS 11b) and multiple depositional sequences.The Cenozoic contains a world class hydrocarbon province with prolific oil reservoirs in the Oligo‐Miocene Asmari Formation sealed by the evaporite‐dominated Gachsaran Formation, mostly contained within giant NW‐SE trending “whaleback” anticlines concentrated in the Dezful Embayment. Reservoirs in the SW are dominantly siliciclastic or comprise mixed siliciclastics and carbonates, whereas those to the east and NE are dominated by fractured carbonates. There remains untested potential in stratigraphic traps, especially in deeperwater sandstone reservoirs deposited along the SW margin of the foredeep.Late Miocene to Pliocene charge to the Asmari reservoirs was mostly from Aptian – Albian Kazhdumi Formation source rocks. In some fields, an additional component was from organic‐rich late Eocene to earliest Oligocene Pabdeh Formation source rocks confined to the narrowing Lurestan – Khuzestan Trough. Where mature, the latter source rock is also a potential unconventional reservoir target, although the prospective area is limited due to recent uplift and erosion. Deeper Jurassic source rocks contributed to the Cheshmeh Khush field in Dezful North. Silurian source rocks charged gas‐bearing structures in the Bandar Abbas region.

Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.

Dihydroguaiazulenide Complexes and Catalysts of Group 8-12 Transition Metals: Ligands from Renewable Feedstock Replace, even Outmatch Petrochemical Based Cyclopentadienyl Chemistry.

We present an in-depth study of the sterically demanding Cp-synthon (8-H-GuaH)Li isolated from natural product guaiazulene (Gua) as a ligand transfer reagent towards late transition metal complex precursors. The synthesis and full characterization of selected, essentially unexplored homo- and heteroleptic 8-H-guaiazulenide complexes of iron, ruthenium, cobalt, rhodium, platinum, copper and zinc are discussed in detail. In order to demonstrate their potential in catalytic applications, [(GuaH)PtMe3 ] was selected. The latter proved an even higher catalytic activity in light induced olefin hydrosilylation at catalyst loads as low as 5 ppm than classical [CpPtMe3 ] in a typical test reaction of silicone elastomer fabrication. Our results demonstrate that traditional petrochemical based Cp metal chemistry and catalysis can be replaced, sometimes even outmatched by superior catalysts based on cheap building blocks from renewable feedstock.

XCR1 expression distinguishes human conventional dendritic cell type 1 with full effector functions from their immediate precursors

Dendritic cells (DCs) are major regulators of innate and adaptive immune responses. DCs can be classified into plasmacytoid DCs and conventional DCs (cDCs) type 1 and 2. Murine and human cDC1 share the mRNA expression of XCR1. Murine studies indicated a specific role of the XCR1-XCL1 axis in the induction of immune responses. Here, we describe that human cDC1 can be distinguished into XCR1- and XCR1+ cDC1 in lymphoid as well as nonlymphoid tissues. Steady-state XCR1+ cDC1 display a preactivated phenotype compared to XCR1- cDC1. Upon stimulation, XCR1+ cDC1, but not XCR1- cDC1, secreted high levels of inflammatory cytokines as well as chemokines. This was associated with enhanced activation of NK cells mediated by XCR1+ cDC1. Moreover, XCR1+ cDC1 excelled in inhibiting replication of Influenza A virus. Further, under DC differentiation conditions, XCR1- cDC1 developed into XCR1+ cDC1. After acquisition of XCR1 expression, XCR1- cDC1 secreted comparable level of inflammatory cytokines. Thus, XCR1 is a marker of terminally differentiated cDC1 that licenses the antiviral effector functions of human cDC1, while XCR1- cDC1 seem to represent a late immediate precursor of cDC1.

Quantitative Assessment of Vacuolization of Myeloid Precursors in VEXAS Syndrome.

Quantitative Assessment of Vacuolization of Myeloid Precursors in VEXAS Syndrome.

Luspatercept (RAP-536) modulates oxidative stress without affecting mutation burden in myelodysplastic syndromes.

In low-risk myelodysplastic syndrome (LR-MDS), erythropoietin (EPO) is widely used for the treatment of chronic anemia. However, initial response to EPO has time-limited effects. Luspatercept reduces red blood cell transfusion dependence in LR-MDS patients. Here, we investigated the molecular action of luspatercept (RAP-536) in an in vitro model of erythroid differentiation of MDS, and also in a in vivo PDX murine model with primary samples of MDS patients carrying or not SF3B1 mutation. In our in vitro model, RAP-536 promotes erythroid proliferation by increasing the number of cycling cells without any impact on apoptosis rates. RAP-536 promoted late erythroid precursor maturation while decreasing intracellular reactive oxygen species level. RNA sequencing of erythroid progenitors obtained under RAP-536 treatment showed an enrichment of genes implicated in positive regulation of response to oxidative stress and erythroid differentiation. In our PDX model, RAP-536 induces a higher hemoglobin level. RAP-536 did not modify variant allele frequencies in vitro and did not have any effect against leukemic burden in our PDX model. These results suggest that RAP-536 promotes in vivo and in vitro erythroid cell differentiation by decreasing ROS level without any remarkable impact on iron homeostasis and on mutated allele burden.

Effect of Denosumab on the Change of Osteoclast Precursors Compared to Zoledronate Treatment in Postmenopausal Women with Osteoporosis.

BackgroundA rapid increase in bone turnover and bone loss has been observed in response to the discontinuation of denosumab. It led to an acute increase in the fracture risk, similar to that observed in the untreated patients. We aimed to investigate the effect of denosumab on osteoclast (OC) precursor cells compared to that of zoledronate.MethodsThe study compared the effects of denosumab (60 mg/24-week) and zoledronate (5 mg/48-week) over 48 weeks in postmenopausal women with osteoporosis. From patients’ peripheral mononuclear cells, CD14+/CD11b+/vitronectin receptor (VNR)- and CD14+/CD11b+/VNR+ cells were isolated using fluorescent-activated cell sorting, representing early and late OC precursors, respectively. The primary endpoint was the changes in OC precursors after 48 weeks of treatment.ResultsAmong the 23 patients, 11 were assigned to the denosumab group and 12 to the zoledronate group (mean age, 69 years). After 48 weeks, the changes in OC precursors were similar between and within the groups. Serum C-terminal telopeptide of type I collagen levels were inversely correlated with OC precursor levels after denosumab treatment (r=−0.72, P<0.001). Lumbar spine, femur neck, and total hip bone mineral density (BMD) increased in both groups. Lumbar spine BMD increased more significantly in the denosumab group than in the zoledronate group.ConclusionsDenosumab and zoledronate treatments induced similar changes in OC precursors. During denosumab treatment, old age and suppressed bone turnover were associated with increased OC precursor cell populations. Further validation studies with prospective designs are required.

The order of immunoglobulin light chain κ and λ usage in primary and secondary lymphoid tissues of germ-free and conventional piglets

In pigs (Sus scrofa), the initial immunoglobulin rearrangement of the κ light chain is replaced by λ before the heavy chains rearrange, and the light chains may rearrange even later. This study investigates whether these developmental differences are reflected in the usage of IGK and IGL genes. We found large differences between peripheral B cells and those developing in the bone marrow, and between B cells in germ-free piglets and conventional pigs. During early B cell development in the bone marrow, more 3′ V and 5′ J gene segments for both light chains are used. However, in the peripheral naive repertoire, more 5′ IGLV and 3′ IGLJ genes are used. A similar shift toward the use of more 5′ IGKV and 3′ IGKJ genes is observed later after antigen exposure in conventional pigs. The expression profile showed that most λ+ B cells are generated earlier, while κ+ B cells develop from late precursors that already contain the λ rearrangement. The initial λ rearrangement is retained in both λ+ and κ+ B lymphocytes, and multiple λ transcripts can be found in individual cells. The overall pool of the IGLV repertoire is therefore much larger and more diversified than for IGKV. The κ repertoire is further restricted to the preferential use of only two major IGKV genes, reflecting the limitation for only two consecutive rearrangements. Tracing of silenced λ transcripts in κ+ B cells further confirmed the unconventional mechanism of differential rearrangements in pigs. Our results underline the diversity of the immune system among mammals.

Unsupervised cluster analysis and subset characterization of abnormal erythropoiesis using the bioinformatic Flow-Self Organizing Maps algorithm.

BackgroundThe Flow‐Self Organizing Maps (FlowSOM) artificial intelligence (AI) program, available within the Bioconductor open‐source R‐project, allows for an unsupervised visualization and interpretation of multiparameter flow cytometry (MFC) data.MethodsApplied to a reference merged file from 11 normal bone marrows (BM) analyzed with an MFC panel targeting erythropoiesis, FlowSOM allowed to identify six subpopulations of erythropoietic precursors (EPs). In order to find out how this program would help in the characterization of abnormalities in erythropoiesis, MFC data from list‐mode files of 16 patients (5 with non‐clonal anemia and 11 with myelodysplastic syndrome [MDS] at diagnosis) were analyzed.ResultsUnsupervised FlowSOM analysis identified 18 additional subsets of EPs not present in the merged normal BM samples. Most of them involved subtle unexpected and previously unreported modifications in CD36 and/or CD71 antigen expression and in side scatter characteristics. Three patterns were observed in MDS patient samples: i) EPs with decreased proliferation and abnormal proliferating precursors, ii) EPs with a normal proliferating fraction and maturation defects in late precursors, and iii) EPs with a reduced erythropoietic fraction but mostly normal patterns suggesting that erythropoiesis was less affected. Additionally, analysis of sequential samples from an MDS patient under treatment showed a decrease of abnormal subsets after azacytidine treatment and near normalization after allogeneic hematopoietic stem‐cell transplantation.ConclusionUnsupervised clustering analysis of MFC data discloses subtle alterations in erythropoiesis not detectable by cytology nor FCM supervised analysis. This novel AI analytical approach sheds some new light on the pathophysiology of these conditions.

In vitro and in vivo detection of tunneling nanotubes in normal and pathological osteoclastogenesis involving osteoclast fusion

AbstractOsteoclasts are multinucleated cells formed through specific recognition and fusion of mononuclear osteoclast precursors derived from hematopoietic stem cells. Detailed cellular events concerning cell fusion in osteoclast differentiation remain ambiguous. Tunneling nanotubes (TNTs), actin-based membrane structures, play an important role in intercellular communication between cells. We have previously reported the presence of TNTs in the fusion process of osteoclastogenesis. Here we analyzed morphological details of TNTs using scanning electron microscopy. The osteoclast precursor cell line RAW-D was stimulated to form osteoclast-like cells, and morphological details in the appearance of TNTs were extensively analyzed. Osteoclast-like cells could be classified into three types; early osteoclast precursors, late osteoclast precursors, and multinucleated osteoclast-like cells based on the morphological characteristics. TNTs were frequently observed among these three types of cells. TNTs could be classified into thin, medium, and thick TNTs based on the diameter and length. The shapes of TNTs were dynamically changed from thin to thick. Among them, medium TNTs were often observed between two remote cells, in which side branches attached to the culture substrates and beaded bulge-like structures were often observed. Cell-cell interaction through TNTs contributed to cell migration and rapid transport of information between cells. TNTs were shown to be involved in cell-cell fusion between osteoclast precursors and multinucleated osteoclast-like cells, in which movement of membrane vesicles and nuclei was observed. Formation of TNTs was also confirmed in primary cultures of osteoclasts. Furthermore, we have successfully detected TNTs formed between osteoclasts observed in the bone destruction sites of arthritic rats. Thus, formation of TNTs may be important for the differentiation of osteoclasts both in vitro and in vivo. TNTs could be one target cellular structure for the regulation of osteoclast differentiation and function in bone diseases.

Multipotent Mesenchymal Stromal Cells from the Bone Marrow of Untreated Aplastic Anemia Patients Preserve Their Ability to Support Hematopoietic Precursors However Display the Pronounced Changes in Gene Expression

Aplastic anemia (AA) is believed to be an autoimmune disorder characterized by the pancytopenia due to the depletion of hematopoietic stem and progenitor cells in the bone marrow. There are three forms of AA depending on the severity of pancytopenia: moderate, or non-severe AA (NAA), severe AA (SAA), and very severe AA (VSAA). Clones of cells typical for paroxysmal nocturnal hemoglobinuria (PNH-clones) are frequently present in AA patients in various proportions.We aimed to study stromal microenvironment of untreated AA patients depending on the AA severity, presence or absence of PNH-clone, and on the response to the therapy after 3 and 6 months of treatment. We analyzed the bone marrow (BM) multipotent mesenchymal stromal cells (MMSCs) in their ability to maintain hematopoietic precursors and examined relative expression levels (REL) of selected genes.The study included 17 patients with NAA (53% females, 47% males, 33.8±2.2 years old), 12 patients with SAA (33% females, 67% males, 29.3±3.8 years old). Among NAA patients 7 had PNH-clone, and among SAA - 6 patients. Control group consisted of 19 donors (42% females, 58% males, 30.4±3.1 years old). The ability to support hematopoietic precursors by MMSCs from the BM of AA patients was measured by cobble stone area forming cells (CAFC) assay, where BM cells from one healthy donor were seeded on different MMSCs; REL of selected genes was analyzed with TaqMan RT-PCR. Only the genes with statistically significant differences are presented. The data are presented as mean ± standard error of measures, the differences were statistically significant when p<0.05 when Student's unpaired t-test or Mann-Whitney test was applied.MMSCs from AA patients preserve their ability to maintain hematopoietic precursors. CAFC 7 frequency reflects the number of late hematopoietic precursors. CAFC 7 frequency was slightly higher on MMSCs from NAA patients (9.92±2.73 per 10 6 healthy BM cells) then on MMSCs from healthy donors (5.56±1.14 per 10 6 healthy BM cells), although the difference was not statistically significant. MMSCs from SAA patients maintained CAFC 7 as well as donors' MMSCs (6.75±1.96 per 10 6 healthy BM cells). The frequency of CAFC 28, reflecting the number of early hematopoietic precursor, displayed similar but more pronounced dynamics. CAFC 28 frequency on NAA patients' MMSCs was significantly higher than on donors' ones (2.17±0.34 versus 1.11±0.31 per 10 6 healthy BM cells, p=0.03), while on SAA patients' MMSCs it was also high (1.92±0.57 per 10 6 healthy BM cells) but the difference was insignificant (Table 1). The presence of PNH-clone does not affect the ability of stromal cells to maintain hematopoiesis. MMSCs from the patients that had responded to the therapy in 90 or 180 days did not differ in their ability to maintain hematopoietic precursors from the MMSCs of treatment resistant the patients. Therefore, we can assume that physiological function of stromal microenvironment is not affected deeply in the debut of AA.Gene expression analysis revealed statistically significant upregulation of FGFR1, PDGFRA, VEGFA and downregulation of ANG1 (in MMSCs from both NAA and SAA patients), and upregulation of FGFR2 and CFH (only in NAA patients' MMSCs) (Table 2). In MMSCs of AA patients (both NAA and SAA) without PNH-clone the upregulation of CFH gene was detected (Table 3). CFH is one of the players in the complement system which is disrupted in PNH. This fact needs to be further scrutinized. In addition, IL1R, SDF1 and VEGFA were statistically significantly downregulated in MMSCs from AA patients with PNH-clone compared with MMSCs from patients without PNH-clone. It seems that the presence of PNH-clone corresponds with the changes in stromal microenvironment. Gene expression of analyzed genes was the same in MMSCs of the patients that had responded or not responded to the treatment in 90 or 180 days since the therapy begun.Thus, MMSCs from the BM of untreated AA patients preserve their ability to support hematopoietic precursors however display the pronounced changes in gene expression.The work is supported by the RFBR, project 19-015-00280. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An Inducible Leukemia-Associated Transcription Factor Facilitates Large-Scale Ex Vivo Generation of Functional Human Macrophages

Long-term ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) proves to be unfeasible as cellular differentiation occurs when HSPCs are detached from their supporting bone marrow stem cell niche. This issue renders it difficult to make use of the proliferation capacity of HSPCs to subsequently produce functional blood cells in relevant numbers, e.g. for cell therapy approaches. To circumvent this challenge, leukemia-associated chimeric transcription factors, including MLL fusion proteins, can be exploited for their pronounced ability to propel cell proliferation while preserving cell immaturity. By designing the protein's activity controllable, the immature state can be abolished at an arbitrary point in time enabling terminal differentiation.In this study, we employed the fusion gene mixed lineage leukemia/eleven nineteen leukemia (MLL-ENL) for engineering an inducible protein switch. For this purpose, we fused the coding sequence of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the transcription factor MLL-ENL and subsequently expressed the protein switch (DD-MLL-ENL) in human CD34+ HSPCs derived from adult healthy donors. In the presence of the specific ligand Shield1, DD-mediated protein degradation is prevented leading to massive and long-term expansion of HSPC-derived late monocytic precursors in the presence of IL-3, IL-6, SCF, FLT3-L, TPO and GM-CSF. The cells do not exhibit additional driver mutations, feature a normal karyotype and telomere length, and sustain immaturity that is strictly dependent on Shield1 supplementation every other day even after two years of ex vivo culture. Upon Shield1 deprivation, the cells completely lost self-renewal and colony-forming properties and spontaneously differentiated. By changing the cytokines to GM-CSF in combination with IFN-γ and LPS we differentiated the progenitor cells into macrophages (MΦ) (Fig. 1 A, B). Immunophenotypic characterization revealed upregulation of the monocyte/macrophage-associated surface markers CD14, CD80, CD86, CD163 and MHC class I and II, concordant with monocytic morphology as judged by cytospin preparations. Analysis of the transcription of selected inflammatory genes, including IL-6 and IL-10, revealed overlapping M1 and M2 macrophage characteristics. Furthermore, mRNA expression profiles using nCounter Systems technology covering a total of 770 myeloid innate immunity-related genes proves the cells' identity as differentiated phagocytes shown by upregulation of gene clusters involved in Fc receptor signaling, TLR signaling, antigen presentation and T cell activation. In functional assays, we demonstrated the ability of the obtained cells to migrate towards the chemokine CCL2 in a 3D chemotaxis assay, attach to VCAM-1 under flow and shear stress and produce reactive oxygen species. Regarding the cells' phagocytic capability, we could verify the uptake of bacterial particles as well as apoptotic cells in efferocytosis assays. Finally, we demonstrated IgG Fc region recognition and binding by the expressed Fcγ receptors enabling phagocytosis of lymphoblastic tumor cells, including Daudi, Raji and patient-derived MCL cells in an antibody-dependent manner using rituximab (RTX), daratumumab (Dara) and trastuzumab (Trast) as a negative control (Fig. 1C).Overall, we could demonstrate the conversion of a harmful leukemic transcription factor into a useful molecular tool for large-scale ex vivo production of functional blood cells. Such engineered controllable protein switches might have the potential to be employed as molecular tools to produce functional immune cells for cell-based immunotherapeutic approaches. [Display omitted] DisclosuresRedondo Monte: Minaris Regenerative Medicine: Current Employment. Beier: Alexion: Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Other: Travel reembursement. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. Greif: AstraZeneca: Honoraria.

Regulation of Iron Homeostasis and Efficacy of Rusfertide Analog Peptide in a Mouse Model for Polycythemia Vera

Polycythemia Vera (PV) is a rare blood disease where mutations in JAK2 kinase confer constitutive JAK2 activity leading to abnormally elevated erythropoiesis that is independent of erythropoietin. PV patients present with iron deficiency at diagnosis due to increased iron utilization for erythropoiesis (Ginzburg YZ, Leukemia 2018) which worsens after repeated therapeutic phlebotomy (TP) performed to maintain hematocrit below 45%. The resulting suppression of hepcidin, the body's main negative regulator of iron metabolism, fuels expanded erythropoiesis resulting in a continued need for TP and thereby exacerbating patients' iron deficiency.Rusfertide targets iron exporter membrane protein ferroportin to trigger its degradation, preventing iron export from cells responsible for dietary iron absorption and cells that store and recycle iron. The resulting pharmacodynamic effect of lowered serum iron has disease-modifying effects in PV (Ginzburg YZ, Leukemia 2018). Rusfertide essentially eliminated the need for therapeutic phlebotomy in all PV patients (Kremyanskaya M, Blood 2020 136 Suppl 1: 33). Rusfertide also reversed iron deficiency, as indicated by increased serum ferritin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) in these patients.We present results from studies in a mouse PV model with JAK2-V617F mutation as in human PV (Mullaly A, Cancer Cell 2010; JAX stock #031658). We show that rusfertide analog Peptide A is efficacious in lowering hematocrit (HCT) while modulating other hematological parameters. Further, we show redistribution of iron away from erythropoiesis and renormalization of iron homeostasis as evidenced by ferrokinetic parameters.PV mice were treated over 6-weeks (thrice per week) with Vehicle or Peptide A at 2.5 or 7.5 mg/kg. At the end of 6 weeks, hematology parameters HCT, hemoglobin, RBC counts, were elevated in the PV-Vehicle group as compared to wild type (WT-Vehicle) mice (Table 1). Hematology parameters in PV-2.5 mg/kg group were lowered to WT-Vehicle values. In PV-7.5 mg/kg group, these parameters were lower than WT-Vehicle values, indicating that excessive iron restriction (EIR) leads to the expected anemic conditions. MCH and mean corpuscular hemoglobin concentration (MCHC) in PV-Vehicle group and PV-2.5 mg/kg treated were comparable to WT-Vehicle, indicating a lack of EIR. For the PV-7.5 mg/kg treated group, MCH and MCHC were significantly lower than WT-Vehicle, suggesting EIR at a high dose impacts hemoglobin concentration of RBC.To investigate the impact of iron restriction with Peptide A on erythroblast precursor cells in bone marrow, we conducted flow-cytometry analysis by gating on CD71 and TER-119 expression, and measuring intracellular iron using Ferro Far Red (FFR) dye. The CD71 + early precursor cell population did not change with Peptide A treatment however, the CD71 -/TER-119 + late precursor cell population was significantly lowered (~4-fold and 7.5-fold, in 2.5 and 7.5 mg/kg Peptide A treated PV groups respectively). Iron levels of CD71 + cells were dose-dependently and statistically significantly reduced in the Peptide A treated groups as compared to PV-Vehicle group. Iron levels of CD71 - cells were marginally lowered only in the PV-7.5 mg/kg group.We investigated the nature of iron redistribution induced by Peptide A, by using flow assay to assess iron concentration in splenic macrophages (F4/80 +/CD11b +). Iron was ~2-fold higher in the PV-7.5 mg/kg group as compared to PV-vehicle, and marginally higher in PV-2.5 mg/kg group. Total tissue iron concentration in the spleen was elevated in a dose-related manner in Peptide A treated groups compared to PV-Vehicle group, and in commensuration serum ferritin was increased. Serum iron was ~2-fold lower in PV-Vehicle group as compared to WT-Vehicle indicating iron depletion due to increased iron utilization for erythropoiesis. Serum iron measured after clearance of Peptide A from circulation (48 hr post-dose), was marginally increased for both Peptide A treated groups compared to PV-Vehicle.These data demonstrate that treatment with rusfertide and analogs, restricts iron from erythropoiesis by sequestering it in macrophage storage compartments. These effects along with normalization of iron homeostasis contribute to usefulness of rusfertide dose titration treatment in maintaining HCT <45% and improving symptoms related to iron deficiency in human PV. [Display omitted] DisclosuresTaranath: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhao: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Vengalam: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tang: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dion: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Su: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tovera: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhandari: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cheng: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mattheakis: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company.

MDS-223: Response to Erythroid Stimulating Agents (ESA) and Flow Cytometric Characteristics of Bone Marrow Erythroid Precursors in IPSS-R Lower-Risk Myelodysplastic Syndrome Patients

Context: Standard treatment of anemia in lower-risk myelodysplastic syndromes (LR-MDS) is based on erythropoietic-stimulating agents (ESAs), but some patients are primary refractory (PR) or lose response (secondary refractory, SR). High serum erythropoietin (sEPO) is the main negative predictor, and treatment of ESA-resistant anemia is challenging, so more predictors are needed. EPO promotes differentiation of committed erythroid cells (COMe) into erythroid precursors (EP), constituted by early (EEP) and late (LEP) precursors. Flow cytometry (FC) can characterize COMe and EP. RED score is an FC parameter that assesses dyserythropoiesis. Objective: We aimed to correlate COMe, EP, and RED score with response to ESAs, WHO category, and sEPO in 44 anemic LR-MDS patients. Design: We evaluated 10 negative controls, 17 PR patients, 17 SR patients, and 10 long responders (LR, response duration >24 months). Fifty-nine percent had sEPO 500 mU/mL(H-EPO); 23% had single lineage dysplasia (MDS-SLD), 34% multilineage dysplasia (MDS-MLD), 23% ring sideroblasts (MDS-RS), and 20% deletion of 5q (MDS-5q-). FC analysis was performed on bone marrow aspirates for all subgroups at diagnosis and within SR subgroup at loss of response to ESA (SRII). We used anti-CD34, CD36, CD105, CD117, CD33, CD71, and CD45 antibodies to identify COMe, total erythroid cells (TOTe), and EP. Coefficients of variance of CD71 and CD36 were used to calculate RED score. Results: TOTe and COMe were increased in LR-MDS compared to controls (p=0.013 and p=0.04, respectively), with the exception of MDS-5q- cases. In the PR subgroup, LEP were increased and COMe were reduced compared to LR. In the SRII group, COMe were lower than at diagnosis (p=0.039). RED score was higher in PR compared to responders (p=0.011). MDS-5q- cases showed reduced TOTe (p=0.047), while MDS-RS cases had higher LEP and RED score (p=0.008 and p=0.012, respectively). Finally, LEP were increased in the H-EPO group compared to L-EPO (p=0.023). Conclusions: These data suggest that COMe seem to be positively associated with ESA response, whereas higher LEP and RED score are negatively associated and also correlate with MDS-RS and H-EPO. Finally, MDS-5q- cases displayed fewer TOTe and COMe, suggesting premature apoptosis of erythroid precursors.

Entrepreneurial Career Probabilities of Adolescents

Introduction: The extent of new enterprise creation is a key driver contributing to economic, social, individual, and cultural values. Given a relatively low rate of Total Early-stage Entrepreneurial Activity (TEA) in Germany, an understanding of the predictors of adolescent entrepreneurial career preferences is critical in developing ways to foster the interest of young people in entrepreneurship. Although the late precursors of the intention to become self-employed are largely understood, only a few studies have investigated which early individual-level factors affect the subjective probability of becoming an entrepreneur. Objective: The objective of the current study is to identify and statistically examine personality factors that affect the subjective probability of adolescents becoming entrepreneurs. Methods: Based on the German Socio-Economic Panel, we employed logistic regression to research the dependence of the variable “probability of becoming self-employed” on independent variables such as gender, locus of control (LoC), and personality traits for German adolescents aged between 16–17 years. Results: The study reveals a positive influence of the personality traits conscientiousness, extraversion, and LoC on the probability of being self-employed for German adolescents aged between 16–17 years. Agreeableness and neuroticism were found to have no significant effect on the subjective probability of adolescents becoming entrepreneurs, and openness was found to have no significant impact on high likelihood of being self-employed. For adolescents, being female has a significant impact only on a medium probability to be self-employed. Conclusion: To the current body of personality models explaining early adolescent entrepreneurial career preferences, we contribute a model which refers to a representative sample of adolescents in German society.

AQP0 is a novel surface marker for deciphering abnormal erythropoiesis

BackgroundHematopoiesis occurs in the bone marrow, producing a complete spectrum of blood cells to maintain homeostasis. In addition to light microscopy, chromosome analysis, and polymerase chain reaction, flow cytometry is a feasible and fast method for quantitatively analyzing hematological diseases. However, because sufficient specific cell markers are scarce, dyserythropoietic diseases are challenging to identify through flow cytometry.MethodsBone marrow samples from C57BL/B6 mice and one healthy donor were analyzed using traditional two-marker (CD71 and glycophorin A) flow cytometry analysis. After cell sorting, the gene expressions of membrane proteins in early and late erythropoiesis precursors and in nonerythroid cells were characterized using microarray analysis.ResultsAmong characterized gene candidates, aquaporin 0 (AQP0) expressed as a surface protein in early- and late-stage erythropoiesis precursors and was not expressed on nonerythroid cells. With the help of AQP0 staining, we could define up to five stages of erythropoiesis in both mouse and human bone marrow using flow cytometry. In addition, because patients with dyserythropoiesis generally exhibited a reduced population of APQ0high cells relative to healthy participants, the analysis results also suggested that the levels of APQ0high cells in early erythropoiesis serve as a novel biomarker that distinguishes normal from dysregulated erythropoiesis.ConclusionsAQP0 was successfully demonstrated to be a marker of erythroid differentiation. The expression levels of AQP0 are downregulated in patients with dyserythropoiesis, indicating a critical role of AQP0 in erythropoiesis. Accordingly, the level of AQP0high in early erythroid precursor cells may serve as a reference parameter for diagnosing diseases associated with dyserythropoiesis.

Active hematopoiesis triggers exosomal release of PRDX2 that promotes osteoclast formation

Hematopoietic disorders, particularly hemolytic anemias, commonly lead to bone loss. We have previously reported that actively proliferating cancer cells stimulate osteoclastogenesis from late precursors in a RANKL‐independent manner. We theorized that cancer cells exploit the physiological role of bone resorption to support expanding hematopoietic bone marrow and examined if hematopoietic cells can trigger osteoclastogenesis. Using phlebotomy‐induced acute anemia in mice, we found strong correlation between augmented erythropoiesis and increased osteoclastogenesis. Conditioned medium (CM) from K562 erythroleukemia cells and primary mouse erythroblasts stimulated osteoclastogenesis when added to RANKL‐primed precursors from mouse bone marrow or RAW264.7 cells. Using immunoblotting and mass spectrometry, PRDX2 was identified as a factor produced by erythroid cells in vitro and in vivo. PRDX2 was detected in K562‐derived exosomes, and inhibiting exosomal release significantly decreased the osteoclastogenic capacity of K562 CM. Recombinant PRDX2 induced osteoclast formation from RANKL‐primed primary or RAW 264.7 precursors to levels comparable to achieved with continuous RANKL treatment. Thus, increased bone marrow erythropoiesis secondary to anemia leads to upregulation of PRDX2, which is released in the exosomes and acts to induce osteoclast formation. Increased bone resorption by the osteoclasts expands bone marrow cavity, which likely plays a supporting role to increase blood cell production.

Physiological properties and characteristic reactions of hydroxyurea

As it was mentioned in the previous paper, we observed the mechanism of action the interesting drug, first synthesized back in 1869 for the first time, called Hydroxyurea. A century later, phase I and II trials began to test its safety in humans with solid tumors. It was first approved by the FDA in 1967 for the treatment of neoplastic diseases and is presently approved for the treatment of melanoma, resistant chronic myelocytic leukemia (CML), and recurrent, metastatic testicular and ovarian cancer. Sickle cell disease is a genetic disorder that decreases life expectancy by 25 to 30 years. Individuals are diagnosed with sickle cell disease if they have one of several genotypes that result in at least half of their hemoglobin being hemoglobin S (HbS). Sickle cell anemia refers specifically to the condition associated with homozygosity for the Hb S mutation (Hb SS). Several other hemoglobin mutations, when occurring with an Hb S mutation, cause a similar but often milder disease than sickle cell anemia. In addition to reduced life expectancy, patients with sickle cell disease experience chronic pain and reduced quality of life. Painful crises, also known as vaso-occlusive crises, are the most common reason for emergency department use and hospitalization, and acute chest syndrome is the most common cause of death. Prior to the approval of hydroxyurea for use in sickle cell disease, patients with this condition were treated only with supportive therapies. These measures included penicillin in children to prevent pneumococcal disease, routine immunizations, and hydration and narcotic therapy to treat painful events. Red blood cell transfusions increase the blood’s oxygen carrying capacity and decrease the concentration of cells with abnormal hemoglobin, but chronic transfusion therapy predictably leads to iron overload and alloimmunization. Therapies such as hydroxyurea that raise fetal hemoglobin (Hb F, α2γ2) levels are promising because they effectively lower the concentration of Hb S within a cell, resulting in less polymerization of the abnormal hemoglobin.Hydroxyurea’s efficacy in sickle cell disease is generally attributed to its ability to raise the levels of Hb F in the blood; however, the mechanisms by which it does so are unclear. Early studies suggested that hydroxyurea is cytotoxic to the more rapidly dividing late erythroid precursors, resulting in the recruitment of early erythroid precursors with an increased capacity to produce HbF.

Umbilical cord blood cells for the treatment of preterm white matter injury: Potential effects and treatment options.

Preterm birth is a global public health problem. A large number of preterm infants survive with preterm white matter injury (PWMI), which leads to neurological deficits, and has multifaceted etiology, clinical course, monitoring, and outcomes. The principal upstream insults leading to PWMI initiation are hypoxia-ischemia and infection and/or inflammation and the key target cells are late oligodendrocyte precursor cells. Current PWMI treatments are mainly supportive, and thus have little effect in terms of protecting the immature brain or repairing injury to improve long-term outcomes. Umbilical cord blood (UCB) cells comprise abundant immunomodulatory and stem cells, which have the potential to reduce brain injury, mainly due to anti-inflammatory and immunomodulatory mechanisms, and also through their release of neurotrophic or growth factors to promote endogenous neurogenesis. In this review, we briefly summarize PWMI pathogenesis and pathophysiology, and the specific properties of different cell types in UCB. We further explore the potential mechanism by which UCB can be used to treat PWMI, and discuss the advantages of and potential issues related to UCB cell therapy. Finally, we suggest potential future studies of UCB cell therapy in preterm infants.