Granulocytic Progenitor Cells Research Articles

TLR9 promotes monocytic myeloid-derived suppressor cell induction during JEV infection

Myeloid-derived suppressor cells (MDSCs) are a group of heterologous populations of immature bone marrow cells consisting of progenitor cells of macrophages, dendritic cells and granulocytes. Recent studies have revealed that the accumulation of MDSCs in the mouse spleen plays a pivotal role in suppressing the immune response following JEV infection. However, the mechanisms by which JEV induces MDSCs are poorly understood. Here, it was found that JEV infection induces mitochondrial damage and the release of mitochondrial DNA (mtDNA), which further leads to the activation of TLR9. TLR9 deficiency decreases the M-MDSCs population and their suppressive function both in vitro and in vivo. Moreover, the increase of MHCⅡ expression on antigen-presenting cells and CD28 expression on T cells in TLR9−/− mice was positively correlated with M-MDSCs reduction. Accordingly, the survival rate of TLR9−/− mice dramatically increased after JEV infection. These findings reveal the connections of mitochondrial damage and TLR9 activation to the induction of M-MDSCs during JEV infection.

Hippo Kinase MST1-Mediated Cell Metabolism Reprograms the Homeostasis and Differentiation of Granulocyte Progenitor Cells.

The mechanism of the development of granulocyte progenitor cells into neutrophils under steady-state and pathological conditions remains unclear. In this study, our results showed that with the development of neutrophils from hematopoietic stem cells to mature neutrophils, the expression level of the Hippo kinase MST1 gradually increased. Mst1-specific deficiency in myeloid cells caused neutrophilia, with an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte-macrophage progenitors under steady-state conditions and during Listeria monocytogenes infection. Mechanistically, mTOR and HIF1α signaling are required for regulating the balance between glycolysis and succinate dehydrogenase-mediated oxidative phosphorylation, which is crucial for Mst1-/--induced proliferation of granulocyte-monocyte progenitors, lineage-decision factor C/EBPα expression, and granulopoiesis. HIF1α directly regulated C/EBPα promoter activities. Blocking mTOR and HIF1α or adjusting the balance between glycolysis and succinate dehydrogenase-mediated oxidative phosphorylation reversed the granulopoiesis induced by Mst1-/- under steady-state conditions or infection in mice. Thus, our findings identify a previously unrecognized interplay between Hippo kinase MST1 signaling and mTOR-HIF1α metabolic reprogramming in granulocyte progenitor cells that underlies granulopoiesis.

Abstract 71: Melanoma alters the bone marrow immune composition in an age-related manner

Abstract Melanoma accounts for the most skin cancer-related deaths in the United States. As patients age, anti-tumor responses decline, making age a negative independent prognostic factor of overall survival in melanoma. Despite advances in immunotherapeutic modalities, there remains a subset of patients who do not benefit from these treatments. Understanding cancer and age-related changes to immune cell development in the bone marrow can improve patient outcomes. We hypothesize that during aging, the bone marrow niche is reshaped, driving precursor cells to become immunosuppressive. This ultimately decreases the anti-tumor response to melanoma due to the skewing of hematopoiesis to yield decreased immune function. In order to tackle this hypothesis, flow cytometry was used to analyze immune compositional changes in the bone marrow of mice challenged with melanoma. Our preliminary data suggests that when melanoma is present in the skin, there are changes to the bone marrow niche, such as an increase in total myeloid cells. Specifically, in the bone marrow of aged tumor-bearing mice, we see a decrease in Ly6C+Ly6G+ granulocytic progenitor cells and an increase in Ly6C+Ly6G- monocytic progenitor cells compared to the bone marrow of young tumor-bearing mice. We predict that the secretome of the aged bone marrow niche will impact immune cell development by producing more immature myeloid cells that can travel to the tumor site. We also saw an increase in the proportion of CD4+ T-cells in the aged bone marrow when melanoma is present. Thus, there appears to be a shift in cell populations in the aged bone marrow niche when the mice are burdened with melanoma. As a future direction, we plan to investigate the role of the aged bone marrow niche and its secretome on the effectiveness of immunotherapies, such as anti-PD-1. These insights could lead to improved outcomes for elderly patients with melanoma. Citation Format: Alexis Erasta Carey, Yash Chhabra, Ethan Black, Murilo Ramos Rocha, Mitchell Fane, Vania Wang, Daniel Zabransky, Gloria Bravante, Laura Hueser, Ashani Weeraratna. Melanoma alters the bone marrow immune composition in an age-related manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 71.

Investigation of Neutrophil Extracellular Trap (NET) Induction in Patients with Chronic Idiopathic Neutropenia Bearing Mutations in the Mediterranean Fever (MEFV) Gene

Introduction: Chronic idiopathic neutropenia (CIN) is a disorder characterized by the prolonged and unexplained reduction in the number of circulating neutrophils. The pathophysiology of CIN has been associated with an inflammatory bone marrow microenvironment consisting of activated T-cells with an oligoclonal/monoclonal profile and intermediate CD14bright/CD16+ monocytes that induce the apoptotic death of the granulocytic progenitor cells. To probe further the basis of immune cell activation in CIN we have recently investigated by next generation sequencing (NGS) the frequency of mutations in the MEFV gene encoding pyrin, in a cohort of 50 CIN patients without any signs/symptoms of Familial Mediterranean Fever (FMF). We have found that 9/50 patients displayed variants associated with typical or atypical FMF phenotype in the Greek population implicating exon 10 and/or exon 2 (Skendros P et al. Blood, 138, Sup.1, p3124;2021). Aim:MEFV mutations have been associated with increased neutrophil activation in FMF and other inflammatory diseases, neutrophil entry in autophagy and extrusion of Neutrophil Extracellular Traps (NETs) decorated with the pro-inflammatory cytokine IL-1β. The aim of the current study is to investigate the association between MEFV mutations and NET formation in neutrophils of CIN patients. Patients-Methods: We studied 20 CIN patients (17 females and 3 males) aged 27 to 78 years. Six CIN patients harbored at least one MEFV mutation with potential clinical significance (A744S homo/R202Q homo, I720M hetero, R202Q homo, K965R hetero, A744S hetero, M694V hetero) and 14 were negative for MEFV pathogenic mutations. The MEFV R202Q mutation is located in exon 2 whereas all the rest mutations are located in exon 10. We isolated neutrophils from healthy subjects (n=5), age- and sex- matched with the patients, and co-cultured them with serum from CIN patients. We used the supernatants of the cultures to quantify NETs with Sytox green staining for extracellular DNA and/or ELISA for dsDNA/MPO complexes. We also used the serum-treated neutrophils for the evaluation of NETosis by immunostaining using confocal microscopy. In addition, neutrophils isolated from CIN patients were used to detect their direct response to spontaneous NETosis induction. Statistical analysis was performed with one-way ANOVA adjusting for multiple comparisons. Results: By co-culturing patients' sera with healthy neutrophils obtained from five different healthy donors, we did not identify statistically significant differences in NETosis evaluation between the three study groups i.e. CIN patients with MEFV mutations, CIN patients negative for pathogenic MEFV mutations, and healthy individuals. On an individual basis, the patient harboring the double homozygous MEFV mutations (A744S/R202Q) displayed increased NETosis in immunostaining, Sytox and ELISA evaluation, as compared to MEFV unmutated CIN patients and healthy individuals. In addition, ex vivo culture of patients' neutrophils and microscopic observation gave us the indication that CIN patients, regardless of their MEFV gene variants, present increased NETosis compared to healthy individuals, suggesting an overactivation status of neutrophils in CIN. Conclusions: The neutrophils of CIN patients display increased NETosis irrespectively of the MEFV mutation status and may thus contribute to the inflammatory processes associated with the disease, a field that has not been investigated so far. Although in our study MEFV mutations did not appear to contribute significantly to inflammatory processes through the NETosis mechanism in CIN patients, their involvement in other aspects of immune deregulation such as through the mTOR/autophagy pathway remains to be further explored.

Protein Tyrosine Phosphatase PTPRO Signaling Couples Metabolic States to Control the Development of Granulocyte Progenitor Cells.

Protein tyrosine phosphatase (PTPase) is critically involved in the regulation of hematopoietic stem cell development and differentiation. Roles of novel isolated receptor PTPase PTPRO from bone marrow hematopoietic stem cells in granulopoiesis have not been investigated. PTPRO expression is correlated with granulocytic differentiation, and Ptpro -/- mice developed neutrophilia, with an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte progenitors under steady-state and potentiated innate immune responses against Listeria monocytogenes infection. Mechanistically, mTOR and HIF1α signaling engaged glucose metabolism and initiated a transcriptional program involving the lineage decision factor C/EBPα, which is critically required for the PTPRO deficiency-directed granulopoiesis. Genetic ablation of mTOR or HIF1α or perturbation of glucose metabolism suppresses progenitor expansion, neutrophilia, and higher glycolytic activities by Ptpro -/- In addition, Ptpro -/- upregulated HIF1α regulates the lineage decision factor C/EBPα promoter activities. Thus, our findings identify a previously unrecognized interplay between receptor PTPase PTPRO signaling and mTOR-HIF1α metabolic reprogramming in progenitor cells of granulocytes that underlies granulopoiesis.

Increased Frequency of Mutations in the Gene Responsible for Familial Mediterranean Fever (MEFV) in a Cohort of Patients with Chronic Idiopathic Neutropenia

▪Introduction-Aim: Chronic idiopathic neutropenia (CIN) is a neutrophil disorder characterized by the prolonged and unexplained reduction in the number of peripheral blood (PB) absolute neutrophil counts (ANC). The underlying pathogenesis in CIN implicates the production of proinflammatory cytokines by activated lymphocytes and monocytes that induce excessive apoptotic death of the bone marrow (BM) granulocytic progenitor cells. Clonal hematopoiesis identified by next generation sequencing (NGS) of myeloid genes is found in 11% of CIN patients conferring an increased risk for MDS/AML transformation whereas the non-clonal patients display usually a benign course. The basis for the immune cell activation and proinflammatory cytokine production in CIN remains obscure. Based on previously reported data showing increased frequency of mutations of the MEFV gene encoding pyrin in patients with idiopathic inflammatory conditions other than typical Familial Mediterranean Fever (FMF), we sought to investigate the common MEFV mutations in a cohort of well characterized CIN patients.Patients-Methods: We have studied 50 patients fulfilling the previously reported diagnostic criteria of CIN (median ANC 1.5x10 9/L, range 0.2-1.7 x10 9/L), 44 females and 6 males with a median age of 56 years (range 25-87 years) and a long-follow-up (median 132 months, range 8-336 months) in the Department of Hematology of the University Hospital of Heraklion, Crete, Greece. Nonisotopic RNase cleavage assay (NIRCA) analysis was used as first screening method to detect MEFV exons 10 and 2 mutations in DNA extracted from PB or BM samples from CIN patients, confirmed by direct NGS analysis. These sequences contain the main disease-related mutations and polymorphisms.Results: Genetics alterations of MEFV were detected in 22 out of 50 CIN patients (44%). Pathogenic mutations (variants associated with typical or “atypical” FMF phenotype in Greek population) were identified in 10/50 CIN patients (20%). The 20% frequency of MEFV mutations in exon 10 and/or exon 2 in CIN patients is significantly higher compared to the carrier rate of common MEFV mutations in the healthy Greek population (0.7%) according to our previously reported data (P<0.0001, Fisher's exact test). NGS analysis confirmed the mutational pattern of NIRCA and specifically showed: (a) one patient with heterozygous I720M (ATC>ATG; Ile>Met), two patients with heterozygous A744S (GCC>TCC; Ala>Ser) and one with homozygosity, one patient with heterozygous M694V (ATG>GTG; Met>Val), one with heterozygous K695R (AAG>AGG; Lys>Arg) and one with heterozygous M680I (ATG>ATC; Met>Ile), all in exon 10, and (b) four patients with homozygous R202Q mutation in exon 2 (one patient with homozygous A744S co-mutation in exon 10) and two patients with R202Q heterozygosity combined with heterozygosity of I720M and A744S of exons 10, respectively. None of the patients displayed any symptoms/signs of FMF or other systemic inflammatory disease. No statistically significant differences were identified between MEFV mutated and non-mutated CIN patients in the severity of neutropenia or in lymphocyte, monocyte, hemoglobin and platelet counts. A significant difference was identified between the two patient groups in serum IgG (1440±264 vs 1133±245 mg/dl; P = 0.0023, Mann-Whitney test) but not IgA or IgM levels.Discussion: This study reports for the first time that 20% of unselected, consecutive patients with CIN carry mutations of the MEFV gene without clinical manifestations of FMF. Whether these patients represent atypical cases of FMF or the identified MEFV genetic alterations have a pathogenetic/modifying effect in the inflammatory responses associated with CIN is an open/novel field of research. As a first step we are currently investigating the neutrophil autophagic status, IL-1β production and the neutrophil extracellular trap (NET) formation in CIN patients with mutations in MEFV to clarify their potential effect in the immune deregulation known to characterize CIN. DisclosuresNo relevant conflicts of interest to declare.

Germline Runx1 Mutations Induce Inflammation in Hematopoietic Stem and Progenitor Cells and Predispose to Hematologic Malignancies

Patients with Familial Platelet disorder (FPD) have a germline RUNX1 mutation and are at high risk to developing hematologic malignancies (HM), primarily myelodysplastic syndrome and acute myeloid leukemia (lifetime risk~40%). To understand how germline RUNX1 mutations predispose to HM in vivo, we developed a Runx1 R188Q/+ mouse strain , mimicking the FPD-associated R201Q missense mutation.Analysis of the bone marrow cells in Runx1 R188Q/+ mice revealed a significant increase in the total number of bone marrow cells. Immunophenotypic analysis using Sca-1 and Cd86 markers revealed a significant increase in Sca-1 expression in hematopoietic stem and multi-potential progenitor cells, indicating a systemic inflammation in the bone marrow. In addition, the frequency of common-myeloid, granulocytic-monocytic and granulocytic progenitor cells were found significantly increased in the Runx1 R188Q/+ bone marrow. Accordingly, their colony-forming unit capacity was increased when compared to wildtype controls (wt/Runx1 R188Q/+ CFU average = 45/85), indicating a myeloid bias.The number and size of platelets were not altered in Runx1 R188Q/+ mice. However, platelet function was significantly reduced. The activation of the Cd41/Cd61 fibrinogen receptor complex in membrane after thrombin treatment was reduced in Runx1 R188Q/+ platelets. Similarly, the translocation of P-selectin by alpha granules and the secretion of serotonin by the dense granules were also reduced.Hematopoietic progenitor cells isolated from Runx1 R188Q/+ mice revealed a significant reduction in DNA-damage repair response in vitro. Quantitative analysis of nuclei with 53bp1-positive foci in response to ionizing radiation showed a marked increase in 53bp1-positive foci in Runx1 R188Q/+ nuclei, suggesting that Runx1 R188Q/+ cells have a defective repair of double strand DNA breaks. Furthermore, expression of DNA-damage repair pathway-associated Pmaip1 (Noxa) was significantly reduced in irradiated Runx1 R188Q/+ hematopoietic progenitor cells.To understand underlying mechanism responsible for the observed myeloid bias in Runx1 R188Q/+ cells, transcription profiling analysis was performed in myeloid progenitors from wildtype and Runx1 R188Q/+ mice, utilizing RNA-sequencing. A total of 39 genes were significantly deregulated (> 1.5 FC; FDR<0.05), including 8 up- and 31 down-regulated genes. The expression of three repressed genes with important function in hematopoietic differentiation and malignancy (Cdh1, Gja1, and Fcer1a) were validated by qRT-PCR.To study the FPD-associated pre-leukemic process in vivo, wildtype and Runx1 R188Q/+ mice were monitored for 20 months. Although Runx1 R188Q/+ mice remained healthy for 18 months, somatic mutations in their leukocytes were evident at 12 months. Targeted sequencing of 578 cancer genes (mIMPACT panel) in leukocyte DNA of two Runx1 R188Q/+ mice identified somatic mutations in Kdm6a, Setd1b, Amer1, and Esco1 (variant allele frequencies between 0.5% and 2.8%). These mutations were confirmed at stable frequency for eight following months. Since loss of the second Runx1 allele is a frequent somatic event in progression to FPD/HM, we evaluated the predisposition to HM in Mx1Cre-Runx1 R188Q/fl mice over time. Unlike Runx1 R188Q/+ mice, Runx1 R188Q/Δ mice succumbed to myeloid leukemia with a median latency of 37.5 weeks and full penetrance. In addition, the expression of oncogenic Nras-G12D, in Runx1 R188Q/Δ mice reduced the median latency to 14.7 weeks.These studies demonstrate that FPD-associated Runx1 germline mutations induce inflammation in hematopoietic stem cells, induce myeloid expansion with defective DNA-damage response and predispose to HM over time. These studies suggest that anti-inflammatory therapies in pre-symptomatic FPD patients may reduce clonal expansion and predisposition to HM. DisclosuresEbert: Exo Therapeutics: Membership on an entity's Board of Directors or advisory committees; Skyhawk Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Deerfield: Research Funding; GRAIL: Consultancy. Levine: Isoplexis: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Imago: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Lilly: Honoraria; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Incyte: Consultancy; Astellas: Consultancy; Amgen: Honoraria.

Lymphocyte Cytosolic Protein 1 I232F Mutation Impairs Granulocytic Proliferation with a G2/M Block in Severe Neutropenia

▪Introduction: As critical effectors of innate immune response, neutrophils migrate from the vasculature into inflamed tissue, where they engage in phagocytosis and clearance of pathogens and apoptotic cells. Migration, phagocytosis, and granule release require multiple well-coordinated events involving remodeling of the actin cytoskeleton. While mutations of some regulators of actin polymerization, such as Wiskott-Aldrich Syndrome gene (WAS) result in Severe Congenital Neutropenia (SCN), the role of actin cytoskeleton in regulating neutrophil ontogeny and functions remain poorly understood. Lymphocyte Cytosolic Protein 1 (LCP1) is an actin bundling protein encoded by LCP1 gene. A 7-year-old girl presented at age of 2 years with chronic severe neutropenia and frequent infections. Next generation sequencing did not reveal a variant in genes known to cause neutropenia. Whole exome sequencing identified a novel LCP1 p.I232F variant of unknown significance, which was not present in either parent. Bone marrow examination revealed a granulocytic hypoplasia with significant dysmorphic giant granulocytes with complex hypersegmentation and increased apoptosis. (Figure 1A) Based on these observations, we hypothesized that missense mutation in LCP1 (I232F) impairs granulocytic proliferation, and causes neutropenia.Aims: To investigate the role of LCP1 I232F, discovered in a child with severe symptomatic neutropenia, in granulopoiesis.Methods: in vitro experiments involving the expression of wild type and mutant LCP1 I232F in murine myeloblast cell (32D cells) and human cervical cancer HeLa cell. We deployed various biologic and functional studies to investigate the role of LCP1 I232F in granulocyte proliferation, differentiation, survival, and function.Results: To determine the pathophysiologic significance of LCP1 I232F, we stably transduced interleukin 3-dependent murine myeloblast 32D and human cervical cancer HeLa cell lines with a doxycycline-inducible lentiviral constructs containing the cDNA for either wild type LCP1 or LCP1 I232F. The mutant LCP1 I232F expressing 32D cells showed impaired proliferation with the appearance of dysplastic granulocytic cells. (Figure 1A, B) However, we did not observe block in differentiation. A cell cycle arrest at G2/M phase occurred only in the LCP1 I232F expressing cells (Figure 1B) and was associated with an upregulation of markers of cell cycle arrest (Cdkn1a and Tp53). LCP1 I232F overexpression did not lead to increased expression of genes involved in apoptosis or the unfolded protein response. The LCP1 I232F cells with dysplastic features were further analyzed by confocal microscopy, which demonstrated increased level of F-actin and enrichment of F-actin and LCP1 at the cell cortex. Flow cytometric evaluation of these cells further confirmed the presence of increased level of F actin. (Figure 1C) Expression of LCP1 I232F impaired cell motility and invasiveness in both 32D and HeLa cells. However, mutant LCP1 expressing 32D cells demonstrated normal oxidative burst upon treatment with N-formyl-Met-Leu-Phe (fMLP) and phorbol myristate acetate (PMA). Confocal imaging and subcellular fractionation revealed diffuse localization of LCP1, but only the mutant form was found in the nucleus. (Figure 1C) In LCP1, two tandem CH domains (CH1-CH2 and CH3-CH4) formed two actin-binding domains (ABD1 and ABD2) that bind to actin to participate in actin bundling. I232F is located at LCP1-CH1 that interacts with actin. To study how the mutation impacts LCP1-actin binding, we constructed a model of LCP1-ABD1 based on the cryo-EM structure of F-actin/LCP1-ABD2 and performed 40 ns of molecular dynamics (MD) simulations of LCP1-ABD1 and LCP1-ABD1 I232F alone to study their dynamical motions. We found that LCP1-ABD1 I232F adopted a more stable open conformation than LCP1-ABD1 to bind effectively with F-actin. The model implies a prolonged association of LCP1 I232F with actin than wild-type LCP1, and the consequence is the increased stabilization of actin bundles.Conclusions: Like the WAS gain-of-function mutations in SCN, LCP1 I232F resulted in severe neutropenia with dysplastic granulocytic precursors, cell cycle arrest, impaired motility, and increased F actin. We also found decreased proliferation of granulocytic progenitor and precursor cells. Our findings suggest that LCP1 is an important gene involved in granulopoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Frequency and Functional Analysis of Myeloid-Derived Suppressor Cells (MDSCs) in the Peripheral Blood and Bone Marrow of Patients with Chronic Idiopathic Neutropenia (CIN)

Myeloid-derived suppressor cells (MDSCs) are myeloid cells with immunoregulatory properties characterized mainly by suppression of T-cell responses (Bizymi et al, HemaSphere 2019). They are divided in HLA-DRlow/-/CD11b+/CD33+/CD15+ polymorphonuclear (PMN-MDSCs) and HLA-DRlow/-/CD11b+/CD33+/CD14+ monocytic (M-MDSCs) subsets and they are implicated in inflammatory and malignant diseases. Chronic idiopathic neutropenia (CIN), is a (usually benign) neutrophil disorder characterized by persistent and unexplained neutropenia following a detailed clinical/laboratory investigation including anti-neutrophil antibody testing, bone marrow (BM) biopsy and karyotype (Dale &amp; Bolyard, Curr Opin Hematol 2017). Previous studies have shown that neutropenia in CIN is associated with increased apoptosis of BM granulocytic progenitor cells due to an inflammatory BM microenvironment consisting of oligoclonal T-lymphocytes, proinflammatory monocytes and proapoptotic cytokines. The aim of the present study is to explore the possible involvement of the MDSCs in the pathophysiology of CIN by investigating their number in peripheral blood (PB) and BM in association with their functional characteristics. We have studied 100 CIN patients and 49 age- and sex-matched healthy controls. The patients fulfilled the previously described diagnostic criteria for CIN (Papadaki et al, Blood 2003) and had mean neutrophil counts 1095.67 ± 479.52 (median 1215, range 100-1700). MDSC subsets were quantitated by flow cytometry in the PB mononuclear cell (PBMC) fraction using the combination of CD33PC7/CD15PC5/HLA-DRECD/CD14PE/CD11bFITC monoclonal antibodies and the Kaluza analysis software. MDSC subsets were also studied in the BMMC fraction of 24 CIN patients and 8 healthy controls from the study population. The T-cell suppression function of patient MDSCs was evaluated in coculture experiments of immunomagnetically sorted, CFSE stained, normal CD3+ cells with immunomagnetically sorted M-MDSCs and PMN-MDSCs from 4 patients and 4 healthy donors using recombinant human IL-2 as activating factor. CFSE staining was detected in the CD3+ cells on day 0 and day 3 of coculture and analysis was performed with the Fcs Express 7 software. Statistical analysis was performed with the Statistica software. We found that the proportion of PB M-MDSCs was statistically significant lower in CIN patients (1.45% ± 1.82%) compared to controls (3.68% ± 3.12%, Mann-Whitney test, p &amp;lt; 0.0001) (Figure a) whereas the proportion of PB PMN-MDSCs, although lower in patients, did not differ significantly from the controls. The proportion of BM M-MDSCs did not differ significantly between CIN patients and controls whereas the proportion of BM PMN-MDSCs was statistically significant lower in patients (13.27% ± 11.27%) compared to controls (19.49% ± 4.46%; Mann-Whitney test, p = 0.0291) (Figure b). Paired analysis showed that the proportion of PMN-MDSCs were higher in the BMMC compared to PBMC fraction in both CIN patients (13.27% ± 11.27% vs 1.14% ± 1.64%, respectively; Wilcoxon test, p = 0.005) (Figure c) and healthy controls (19.49% ± 4.46% vs 9.92% ± 9.08%, respectively; Wilcoxon test, p = 0.0118). Interestingly, the proportion of increase of PMN-MDSCs (in BMMC vs PBMC fraction) was significantly higher in patients (86.71% ± 21.26%) compared to controls (55.95% ± 38.59%; Mann-Whitney test, p = 0.0357) (Figure d). The above data indicate low production of PMN-MDSCs in CIN patients compared to controls but a trend for accumulation of these cells in patients' BM. No statistically significant difference was documented in paired analysis of M-MDSCs between BMMC and PBMC fractions in either CIN patients or healthy controls. Patient PMN-MDSCs and M-MDSCs displayed normal capacity to suppress T-cell proliferation as was indicated by the T-cell generations in coculture experiments of normal CD3+ cells in the presence or absence of patient MDSCs (Figure e). In conclusion, CIN patients display low proportion of MDSCs in the PB and lower proportion of PMN-MDSC in the BM compared to normal individuals. Patient MDSCs display normal capacity to suppress T-cell activation. The low proportions of MDSCs may sustain the inflammatory process associated with CIN whereas the accumulation of PMN-MDSCs in the BM represents probably a compensatory mechanism to suppress the inflammatory processes within patients' BM microenvironment. Figure Disclosures Papadaki: Genesis pharma SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Protein Arginine Methyltransferase 1 (PRMT1) Dampens Self-Renewal and Promotes Differentiation of Hematopoietic Stem Cells in Mouse Models

Protein arginine methylation is a common type of post-translational modification. PRMT1, the major type I protein arginine methyltransferase, catalyzes the formation of asymmetric dimethyl-arginine and is implicated in various cellular processes, including hematopoiesis and tumorigenesis. We have shown that PRMT1 expression is naturally low in hematopoietic stem cells (HSCs). However, the functions of PRMT1 in hematopoietic stem cell self-renewal and differentiation are yet to be revealed. We have found a cyanine-based fluorescent probe (E84) that can specifically label PRMT1 protein. E84 staining dynamically captures intracellular PRMT1 level and was used to separate live HSC populations with differential PRMT1 expression by flow cytometry. Subsequent bone marrow transplantation of E84high or E84low Lin−Sca1+cKit+ (LSK) cells showed that E84low LSK cells were much more advantageous in reconstituting each blood cell lineages, compared to the E84high counterparts, meaning that the stem-ness of HSCs is negatively correlated with endogenous PRMT1. Therefore, inhibition of PRMT1 was expected to enhance the number and differentiation potential of functional HSCs. The treatment of a PRMT1-specific inhibitor (MS023) to mice resulted in an enlarged LT-HSC population in bone marrow and decreased frequency of granulocyte progenitor cells. In vitro colony formation assays further demonstrated that PRMT1 is required for GMP differentiation. Then we asked whether copious expression of PRMT1 promotes the differentiation of HSC. In this line, we made a LoxP-STOP-LoxP-PRMT1 transgenic mouse model, which induces PRMT1 overexpression upon the expression of Cre recombinase from tissue-specific promoters. We established Mx1-Cre-PRMT1 (Mx1-Tg) mice. Mx1-Tg mice were injected with poly(I:C) for PRMT1 induction and analyzed at four weeks after the last dose. We found that, as predicted, LT-HSC population was reduced and the Pre-GM population was raised. Accordingly, more CFU-Gs but less GEMMs were grown on CFU assays. We further utilized this animal model to compare the blood reconstitution capabilities of bone marrow cells from Mx1-Tg vs. WT mice in the same repopulating conditions. We performed competitive bone marrow transplantation by injecting Mx1-Tg/WT (CD45.2) bone marrow plus supporting cells (CD45.1) to irradiated mice, followed by 5 doses of poly(I:C) induction. Recipient mice were analyzed during a course of approximately 16 weeks. Mx1-Tg cells were outcompeted by WT cells in reconstituting every blood lineages. Taken together, we conclude that PRMT1 promotes HSC differentiation and accelerates HSC exhaustion during the stress caused by bone marrow irradiation. To understand the mechanism on PRMT1-mediated stress hematopoiesis, we also made Pf4-Cre PRMT1 transgenic mice. When PRMT1 is specifically expressed in MK cells, the number of LT-HSCs was also reduced, implying that PRMT1 affects the self-renewal of LT-HSCs via communication between MK cells and HSCs. Mechanistically, two PRMT1 substrates - RBM15 and DUSP4 - are critical for stem cell self-renewal. We further characterized how PRMT1 activates p38 kinase pathway via directly methylating DUSP4 thus induces ubiquitylation and degradation of DUSP4. The arginine methylation site on DUSP4 has been identified. Moreover, introducing methyl-R mutated DUSP4 back to PRMT1-overexpressing cells partially rescued the loss of HSC differentiation potential. This data adds a new link between arginine methylation and protein phosphorylation mediated by MAP kinases/phosphatases. In addition, we discovered that RBM15 controls alternative RNA splicing and RNA processing in a PRMT1-dosage dependent manner. In this report, we will further address how RBM15 target genes, such as enzymes involved in fatty acid metabolic pathways, affect HSC differentiation. In summary, we report that arginine methylation is a novel regulator for the HSC differentiation via controlling p38-regulated stress pathway and metabolic reprogramming. Disclosures No relevant conflicts of interest to declare.

CXCR2 expression on granulocyte and macrophage progenitors under tumor conditions contributes to mo-MDSC generation via SAP18/ERK/STAT3

Myeloid-derived suppressor cells (MDSCs) comprise a critical component of the tumor environment and CXCR2 reportedly plays a key role in the pathophysiology of various inflammatory diseases. Here, CXCR2 expression on granulocyte and macrophage progenitor cells (GMPs) was found to participate in myeloid cell differentiation within the tumor environment. In CXCR2-deficient tumor-bearing mice, GMPs exhibited fewer macrophage and dendritic cell progenitor cells than wild-type tumor-bearing mice, thereby decreasing monocytic MDSCs (mo-MDSCs) expansion. CXCR2 deficiency increased SAP18 expression in tumor-bearing mice, which reduced STAT3 phosphorylation through restraining ERK1/2 activation. Our findings reveal a critical role for CXCR2 in regulating hematopoietic progenitor cell differentiation under tumor conditions, and SAP18 is a key negative regulator in this process. Thus, inhibiting CXCR2 expression may alter the tumor microenvironment and attenuate tumor progression.

Characterization and In Silico Analysis of The Structural Features of G-CSF Derived from Lysates of Escherichia coli.

Objective Granulocyte colony-stimulating factor (G-CSF) has a wide variety of functions including stimulation of hematopoiesis and proliferation of granulocyte progenitor cells. Recombinant human G-CSF (rh-G-CSF) is used for treatment of neutropenia in patients receiving chemotherapy. The mature bloodstream neutrophils express G-CSF receptor (G-CSFR), presenting a significant and specific mechanism for circulating G-CSF clearance. Computational studies are essential bioinformatics methods used for characterization of proteins with regard to their physicochemical properties and 3D configuration, as well as protein–ligand interactions for recombinant drugs. We formerly produced rh-G-CSF in E. coli and showed that the isolated protein had unacceptable biological activity in mice. In the present paper, we aimed to characterize the purified rh-G-CSF by analytical tests and developed an in vivo model by computational modelling of G-CSF. Materials and Methods In this experimental study, we analyzed the purified G-CSF using the analytical experiments. Then, the crystalline structure was extracted from Protein Data Bank (PDB) and molecular dynamics (MD) simulation was performed using Gromacs 5.1 package under an Amber force field. The importance of amino acid contents of G-CSF, to bind the respective receptor was also detected; moreover, the effect of dithiothreitol (DTT) used in G-CSF purification was studied. Results The results revealed that characteristics of the produced recombinant G-CSF were comparable with those of the standard G-CSF and the recombinant G-CSF with the residual amino acid was stable. Also, purification conditions (DTT and existence of extra cysteine) had a significant effect on the stability and functionality of the produced G-CSF. ConclusionExperimental and in silico analyses provided good information regarding the function and characteristics of our recombinant G-CSF which could be useful for industrial researches.

Impairment of G-CSF receptor on granulocytic progenitor cells causes neutropenia in protein malnutrition.

It is well known that protein malnutrition (PM) states can affect hematopoiesis, leading to severe leukopenia and reduced number of granulocytes, which act as the first line of defense, and are important to the innate immune response. The aim of this study was to elucidate some of the mechanisms involved in the impairment of granulopoiesis in PM. Male C57BL/6 mice were submitted to PM with a low-protein diet containing 2% protein. Control mice were fed a 12% protein-containing diet. Bone marrow histology and the percentage of granulocytic progenitors were evaluated after in vivo granulocyte-colony stimulating factor (G-CSF) stimulus. Cell proliferation, STAT3 signaling, and the expression of G-CSF receptor were evaluated in hematopoietic progenitor cells. Malnourished animals presented with leukopenia associated with reduced number of granulocytes and reduced percentage of granulocytic progenitors; however, no differences were observed in the regulatory granulopoietic cytokine G-CSF. Additionally, the malnourished group presented with impaired response to in vivo G-CSF stimulus compared with control animals. PM was implicated in decreased ability of c-Kit+ cells to differentiate into myeloid progenitor cells and downregulated STAT3 signaling. Furthermore, the malnourished group exhibited reduced expression of G-CSF receptor on granule-monocytic progenitors. This reduced expression was not completely reversible with G-CSF treatment. This study implies that PM promotes intrinsic alterations to hematopoietic precursors, which result in hematologic changes, mainly neutropenia, observed in peripheral blood in PM states.

PB1858 THE RESULTS OF THE COLONY‐FORMING ABILITY‘S DETERMINATION OF THE BONE MARROW IN CHILDREN WITH NEUTROPENIA

Background:Primary neutropenia is a rare hematological condition, mostly common in childhood. Neutropenic syndrome is heterogenous and may be a consequence of myelopoietic disorder in bone marrow, redistribution of neutrophils between the wall and circulating cell pools, enhanced destruction of granulocytes, and combination of these mechanisms. In this regard, the options for neutropenic conditions in children are very numerous. Determination of the pathogenetic mechanism of the development of neutropenia is possible only with a deep laboratory examination and is necessary to determine the tactics of patient‘s management. A cultural method of bone marrow's colony‐forming ability‘s assessing is one of the modern diagnostic approaches for cytopenias.Aims:Assessment the significance of the results of the bone marrow colony‐forming ability‘s determining at primary neutropenia in children.Methods:The results of laboratory tests of 10 patients examined for neutropenia were analyzed. The criterion of the diagnosis was considered an absolute decrease in the number of neutrophils in children over the age of 1 year ‐ less than 1,5 thousand in 1 μl of blood. The median of the age of patients was 9.5 (2.5‐17), among them there were 7 boys and 3 girls. To exclude system blood diseases, standard cytological and cytogenetic studies of the bone marrow were performed. The determination of the proliferative potential and differentiation capacity of the bone marrow was carried out by counting the number of granulocyte‐marcophagal‐erythrocyte‐megakaryocytic (CFU‐GEMM, normal 0‐8), granulocyte (CFU‐G, normal 7‐35), macrophage (CFU‐M, normal 1 ‐18), eosinophilic (CFU‐Eoz, norm 0‐22) and erythroid colonies (CFU‐Er, normal 20‐74) in a 14‐day culture. For this purpose, 0.2‐0.4 x 105 myelokaryocytes were introduced into the methylcellulose‐based medium (MethoCult H, StemCell Technologies). The number of formed colonies was considered as equal to the quantity of the corresponding colony‐forming units in the analyzed sample.Results:At the time of the examination, the number of leukocytes in the peripheral blood was 3.4 ± 1.3x103/μl, and the absolute neutrophil content was 969 ± 871.3/μl. Depending on the results of the bone marrow‘s colony‐forming ability patients were divided into 2 groups ‐ with normal and reduced CFU‐G content. In 7 out of 10 patients with neutropenia, a reduced content of granulocyte progenitor cells in the bone marrow was detected: the number of CFU‐G was 2‐6, the number of the remaining CFU was in the normal range (CFU‐GEMM ‐ 0‐5, CFU‐M ‐ 11‐40, CFU‐Er 3‐56, CFU‐Eoz 0‐15). The lowest absolute neutrophil content per unit of peripheral blood‘s volume (170‐1325 in 1 μl, median ‐ 920) was recorded at patients of this group. In case of dynamic observation, infectious complications of various localization and severity were registered, which, along with antibacterial drugs, required the administration of a granulocyte colony‐stimulating factor (G‐CSF). The use of G‐CSF was accompanied by a pronounced positive effect.In the remaining cases (3 out of 10 patients), the bone‐marrow colony‐forming ability was within the normal range (CFU‐G ‐ 12‐36, CFU‐M ‐ 18‐46, CFU‐Er ‐ 10‐56, CFU‐Eoz ‐ 2 ‐ 5). In the peripheral blood of these patients the absolute number of neutrophils was moderately reduced (1000‐1840 in 1 μl, median ‐ 1650). Upon further observation of this patients, indications for G‐CSF and severe infectious diseases were not observed.Summary/Conclusion:Assessment of the results of bone marrow‘s cultural research allows to predict primary neutropenia‘s course and administrate pathogenic therapy.

Comparison of pegfilgrastim (Peg), plinabulin (Plin), and the combination for chemotherapy (Chemo) induced neutropenia (CIN) prevention: Rationale for the combination.

e12030 Background: Peg is used for prevention of CIN. Plin, a novel non-GSF small molecule with anti-cancer activity has equal efficacy against CIN as Peg; Plin does not cause bone pain and mitigates thrombocytopenia and the immune suppressive hematologic profile (Blayney ASCO, ESMO, SITC 2018). Plin has a mechanism of action (MoA) consistent with IL-6 -dependent granulocyte progenitor cell expansion, increased neutrophil demargination and transit from the bone marrow (Suwa, Am J Physiol 2000; Blayney SLB/IEIIS 2018). In single agent (SA) Peg and Plin phase (Ph) 2 data, CIN with Peg occurs in the 1st week (~day (D) 7) post Chemo, and with Plin in the 2nd week (~ D15) post Chemo, presumably due to different MoAs (NCT03102606, NCT03294577). We evaluated whether combining these two agents/MoAs would protect against CIN throughout the entire Chemo cycle (C). Methods: Early stage Breast Cancer Ph 2 patients received high febrile neutropenia (FN) risk TAC (docetaxel, doxorubicin, cyclophosphamide) and either SA standard dose Peg 6 mg (Peg; n=22) or SA Plin (20 mg/m2; n= 15), or Peg 6mg combined with Plin 20 mg/m2 (Peg/Plin; n=16). Peg was given ~24 hrs after TAC, and Plin ~30 min after TAC. Blood draws for ANC were taken daily from predose to Day (D)15, and validated bone pain assessments were done from predose D1 to D8 in C1. Results: On D7 (week 1) of C1, Plin but not Peg maintained mean ANC within normal; P&lt;0.0001 for Plin vs Peg mean ANC. On D15 (week 2 of C1) Peg, but not Plin maintained ANC within normal; P&lt;0.0001 for Peg vs Plin mean ANC. Plin added to Peg maintained mean and median ANC within normal throughout C1. (ANC normal range is 1.5 - 8 x10E9/L.) Frequency of Grade (Gr) 4 and 3/4 CIN was ~35 % lower (P&lt;0.05 for Gr3/4) and median ANC nadir was 2 times higher (1.00 vs 0.46) in Peg/Plin vs Peg in C1. FN occurred in 1 pt each with Peg and Peg/Plin. Incidence of bone pain was 95% vs 6% with Peg vs Peg/Plin (P&lt;0.0001). Conclusions: Adding Plin to standard dose Peg offers superior CIN protection throughout the entire high FN risk Chemo cycle, while almost eradicating bone pain vs Peg alone. Clinical trial information: NCT03294577 .[Table: see text]

CXCR4 Has a CXCL12-Independent Essential Role in MLL-AF9 Driven Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a clonal hematological disorder associated with poor prognosis, and there is a strong need to develop new therapeutic strategies. AML is propagated by a small population of leukemic cells in the bone marrow with self-renewal capacity, a population termed leukemia stem cells (LSC). To identify in vivo dependencies of LSCs, we conducted a CRISPR/Cas9 drop-out screen targeting cell surface receptors on murine LSCs. To this end, we generated a pooled lentiviral CRISPR library targeting 96 cell surface receptors that are upregulated on murine MLL-AF9 LSC compared to normal granulocyte and macrophage progenitor (GMP) cells based on global gene expression data. The CRISPR library was transduced into Cas9-expressing murine c-Kit+MLL-AF9 AML cells and subsequently injected into sublethally irradiated (600 cGy) C57BL/6 mice. Two weeks later when the mice had developed leukemia, the bone marrow was harvested, and sgRNAs sequenced. A high reproducibility in sgRNA representation was observed between five biological replicates, demonstrating that the screen was robust.The screen ranked CXCR4 as the most critical cell surface receptor on AML stem cells with all five sgRNAs targeting Cxcr4 depleting more than 4-fold in vivo. Cxcr4 has previously been shown to be critical in several types of cancers including hematological malignancies. Moreover, CXCR4 has been shown to be important for homing of normal and leukemia stem cells in the bone marrow. To validate the role of CXCR4 in a microenvironment and cell intrinsic manner in AML, we disrupted Cxcr4 in c-Kit+MLL-AF9 AML cells using lentiviral vectors expressing sgRNAs targeting Cxcr4 and GFP as a marker gene. Whereas Cxcr4 disruption did not affect the growth and survival of the AML cells in in vitro cultures with standard cytokines, a 15-fold depletion (p = 0.006) of Cxcr4 sgRNA-expressing AML cells was observed 12 days post-transplantation in vivo. We next studied whether the loss of CXCR4 on the surface of leukemic cells would translate into a survival difference in vivo. Notably, the mice receiving sorted leukemia cells expressing Cxcr4 sgRNAs did not develop AML, demonstrating that CXCR4 is essential for LSCs in vivo. To investigate if this effect was due to a homing defect, we transplanted Cxcr4 sgRNA-expressing cells into sublethally irradiated mice and measured the number of AML cells in the bone marrow after 24 hours. A 2.5-fold reduction (p = 0.03) of leukemia cells expressing Cxcr4 sgRNA compared to the control sgRNA was observed in the bone marrow, suggesting that the depletion of leukemic cells in vivo was partially due to a homing defect.CXCL12, the most studied ligand of CXCR4, is a homeostatic chemokine widely expressed by several cell types in the bone marrow and exists both in a membrane and soluble form. Although the CXCR4-CXCL12 interaction is critical for the retention of normal hematopoietic stem and progenitor cells in the bone marrow, the role of CXCL12 in AML is less characterised. To study the role of different CXCL12-expressing bone marrow cell populations in AML development, we first transplanted c-Kit+MLL-AF9 leukemia cells into Cxcl12f/f-Tek-Cre+ mice, which are devoid of Cxcl12 expression in endothelial cells, and in Cxcl12f/f-Prx1-Cre+ mice, which lack expression of Cxcl12 in mesenchymal stem cells. No reduction in leukemia levels in the blood and bone marrow was observed with either of these mouse strains as recipients, indicating that Cxcl12 expression by endothelial cells and MSC is not critical for AML development. Next, we repeated the experiment using Cxcl12f/f-Ubc-Cre+ mice, which lack Cxcl12 expression in all cells. Intriguingly, there was no reduction in leukemia levels in these mice. Instead, a significant 2-fold increase in leukemia levels in the peripheral blood (p=0.011) and a 1.5-fold increase in leukemia levels in the bone marrow (p=0.043) were observed 12 days after transplantation. These findings suggest that the CXCR4-CXCL12 interaction is dispensable for AML development and progression.Taken together, we here established an in vivo pooled CRISPR/Cas9 drop-out screen using murine LSCs and identified Cxcr4 as an essential regulator for LSCs. Further validations identified that CXCR4 has a previously uncharacterized CXCL12-independent essential role for MLL-AF9 AML cells. Ongoing studies are aimed at identifying the ligand that is essential for CXCR4 in AML. DisclosuresNo relevant conflicts of interest to declare.

Isolation and Culture of Amphibian (Xenopus laevis) Sub-Capsular Liver and Bone Marrow Cells.

The X. laevis sub-capsular liver is thought to be the principal hematopoietic site of Xenopodinae species from early development and, in case of certain species, into adulthood. The Xenopus bone marrow appears to be comprised of precursor cells committed to myeloid lineages, such as macrophage- and granulocyte-progenitor cells. With alarming increases in the contribution of pathogenic infections to the global amphibian declines, now more than ever a better understanding of the mechanisms controlling amphibian immune cell ontogeny and functionality is warranted. Accordingly, here we detail the isolation and culture of the X. laevis hematopoietic cells from the sub-capsular liver and bone marrow. Considering the immunological roles attributed to these amphibian organs, the respective cell isolation protocols described here will be pertinent to garnering further insights into the coordinated regulation of amphibian hematopoiesis and immune defense mechanisms.

Apolipoprotein A-I Stimulates Cell Proliferation in Bone Marrow Cell Culture.

Culturing of bone marrow cells in serum-free RPMI-1640 medium led to a decrease in the rate of DNA biosynthesis. Addition of HDL or their main protein component apolipoprotein A-I to the culture medium dose-dependently increased the rate of [3H]-thymidine incorporation into DNA. The maximum stimulation was achieved at HDL concentration of 80 μg/ml and apolipoprotein A-I concentration of 20 μg/ml. To identify the target-cells of apolipoprotein A-I, we used thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) that incorporates into cell DNA at the stage of replicative DNA synthesis (S phase) and can be detected by fluorescence microscopy. In bone marrow cell culture, apolipoprotein A-I stimulates the proliferation of monocyte (monoblasts, promonocytes) and granulocyte (myeloblasts, promyelocytes) progenitor cells, as well as bone marrow stromal cells.

Ginseng-Derived Panaxadiol Saponins Promote Hematopoiesis Recovery in Cyclophosphamide-Induced Myelosuppressive Mice: Potential Novel Treatment of Chemotherapy-Induced Cytopenias

To investigate the potential efficacy of panaxadiol saponins component (PDS-C), a biologically active fraction isolated from total ginsenosides, to reverse chemotherapy-induced myelosuppression and pancytopenia caused by cyclophamide (CTX). Mice with myelosuppression induced by CTX were treated with PDS-C at a low- (20 mg/kg), moderate- (40 mg/kg), or high-dose (80 mg/kg) for 7 consecutive days. The level of peripheral white blood cell (WBC), neutrophil (NEU) and platelet (PLT) were measured, the histopathology and colony formation were observed, the protein kinase and transcription factors in hematopoietic cells were determined by immunohistochemical staining and Western blot. In response to PDS-C therapy, the peripheral WBC, NEU and PLT counts of CTX-induced myelosuppressed mice were significantly increased in a dose-dependent manner. Similarly, bone marrow histopathology examination showed reversal of CTX-induced myelosuppression with increase in overall bone marrow cellularity and the number of hematopoietic cells (P<0.01). PDS-C also promoted proliferation of granulocytic and megakaryocyte progenitor cells in CTX-treated mice, as evidenced by significantly increase in colony formation units-granulocytes/monocytes and -megakaryocytes (P<0.01). The enhancement of hematopoiesis by PDS-C appears to be mediated by an intracellular signaling pathway, this was evidenced by the up-regulation of phosphorylated mitogen-activated protein kinase (p-MEK) and extracellular signal-regulated kinases (p-ERK), and receptor tyrosine kinase (C-kit) and globin transcription factor 1 (GATA-1) in hematopoietic cells of CTX-treated mice (P<0.05). PDS-C possesses hematopoietic growth factor-like activities that promote proliferation and also possibly differentiation of hematopoietic progenitor cells in myelosuppressed mice, probably mediated by a mechanism involving MEK and ERK protein kinases, and C-kit and GATA-1 transcription factors. PDS-C may potentially be a novel treatment of myelosuppression and pancytopenia caused by chemotherapy.

First description of the rs45496295 polymorphism of the C/EBPE gene in β-thalassemia intermedia patients

The C/EBPE gene, located in 14q11.2, encodes for a B/zip-type transcription factor. The C/EBPɛ is involved in terminal differentiation and functional maturity of granulocyte progenitor cells and in cell apoptosis during myeloid differentiation. A C/EBPE gene has recently been described as a candidate gene involved in clinical variability of β-thalassemia (β-thal). In this study, the C/EBPE gene was sequenced in 146 subjects divided into the severe type of β-thal major (β-TM) and moderate type of β-thal intermedia (β-TI), and a control group. The analysis identified the rs45496295 (C > T) polymorphism in the heterozygous state in 73.9% β-TI patients, which was not the case in the β-TM patients or in the control group. Thus, the T allele is consequently associated with the β-TI group (p = 10−3). According to the Human Splicing Finder (version 3.0, Marseille, France), the presence of the rs45496295 polymorphism leads the creation of a new intronic exotic splicing enhancer (ESE) site. Moreover, the T allele of rs45496295 is associated with a lower transfusion regimen (p = 10−3) and a higher pretransfusion hemoglobin (Hb) rate (p = .006). The comparison of several factors concerning T allele carriers and non-carriers showed that the T allele does not act on the Hb F rate. The T allele of rs45496295, associated with moderate type of β-thal, seems to modify the C/EBPɛ action, thereby preventing the hemolysis.