Erythroid Precursors In Bone Marrow Research Articles

Iron Restriction Alleviates β-Thalassemia By Stimulating ULK1-Mediated Autophagy of Free α-Globin

In β-thalassemia, mutations in the HBB gene cause reduced β-globin synthesis with accumulation of toxic free α-globin, leading to ineffective erythropoiesis and hemolysis. We showed previously that β-thalassemic erythroblasts can eliminate free α-globin by ULK1-mediated autophagy and that this process is stimulated by rapamycin inhibition of mTORC1, which phosphorylates ULK1 to inhibit its activity. Several studies have shown that iron restriction can reduce the accumulation of free α-globin precipitates to alleviate the pathophysiology of β-thalassemia. Considering that iron stimulates mTORC1 activity in numerous cell types including erythroblasts, we tested the hypothesis that iron restriction alleviates β-thalassemia by inhibiting mTORC1, thereby stimulating ULK1-mediated autophagy of free α-globin. Hbb Th3/+ β-thalassemic mice or Hbb Th3/+ Ulk1 -/- mice were treated with the clinical stage oral ferroportin inhibitor vamifeport (known also as VIT-2763) for 45 days to cause systemic iron restriction. In Hbb Th3/+ mice, vamifeport administration led to 66% and 40% reductions in the levels of serum iron (p<0.001) and ferritin (p=0.001), respectively, as expected. Iron restriction by vamifeport alleviated the pathophysiology of β-thalassemia compared to vehicle-treatment in Hbb Th3/+, as evidenced by a 21% increase in RBC count (p<0.001), 32% reduction in insoluble α-globin in red blood cells (RBC) (p=0.002), 41% reduction in reticulocyte count (p<0.001), 27% reduction in spleen mass (p=0.034) and enhanced maturation of spleen and bone marrow erythroid precursors, indicating reduced ineffective erythropoiesis. Multivariate analysis showed that beneficial effects of iron restriction in β-thalassemic mice were attenuated by concomitant disruption of both Ulk1 alleles. For example, vamifeport treatment of Hbb Th3/+ Ulk1 -/- mice caused only a 12% increase in RBC counts (p=0.015 compared to Hbb Th3/+). Additionally, iron restriction improved erythroid maturation in Hbb Th3/+ Ulk1 -/- mice, but to a significantly lesser extent than in thalassemic mice with intact Ulk1alleles. We obtained similar results by inducing iron deprivation with PR73, a peptide hepcidin mimetic. Mechanistically, long-term administration of vamifeport was associated with inhibition of mTORC1, as evidenced by 32% reductions in the phosphorylation of its substrate, ribosomal protein S6 kinase (p=0.004), and activation of the heme-regulated eIF2α kinase (HRI), indicated by increased phosphorylation of eIF2α and/or accumulation of the HRI effector transcription factor ATF4. Similar results were obtained with PR73. Administration of a single dose of PR73 to Hbb Th3/+ mice led to activation of HRI in Ter119 + erythroblasts after 8 hours, which preceded the downregulation of mTORC1 activity, detected at 24 hours. This is consistent with prior studies showing that HRI inhibits mTORC1. Loss of ULK1 had no effect on suppression of mTORC1 with either drug, consistent with ULK1 being the substrate of mTORC1; we are currently investigating whether loss of ULK1 alters the HRI pathway. Together, our data support a mechanism through which iron restriction alleviates the pathophysiology of β-thalassemia in part by stimulating ULK1-mediated autophagy of free α-globin. This process is likely mediated by iron restriction that activates HRI which in turn inhibits mTORC1 to block its negative effects on ULK1. Additional mechanisms for iron regulation of mTORC1 activity are also possible. Our findings provide new insight into the mechanisms by which iron restriction alleviates β-thalassemia and illustrate how metabolic regulators of erythropoiesis impact the severity of β-thalassemia by eliminating free α-globin through a key protein quality control pathway.

Influence of soluble factors from the M2 phenotype macrophages on hematopoiesis in depression-like state

Chronic psychosocial stress provokes anxious behavior and depressive disorders. The longitudinal stress-induced neuroendocrine signals may alter functioning of immune (central and peripheral) organs. Increased myelopoiesis is observed in bone marrow, being detrimental to lympho- and erythropoiesis, with increased emigration of monocytic bone marrow cells to the periphery and their acquisition of “inflammatory” phenotype. The subsequent migration of such monocytes to the brain with differentiation into the M1 type macrophages which form inflammatory signals, and their effect upon endothelial cells and microglia leads to increased production of cytokines, chemokines, and adhesion molecules, thus accelerating accumulation of bone marrow-derived monocytes migrating to the brain. The signals from bone marrow monocytes and activated microglia promote neuroinflammatory condition which leads to behavioral changes. Current data on the presence of non-resident bone marrow macrophages in the brain of depressed patients require studies of hematopoiesis in depression-like states. Pronounced plasticity is a characteristic feature of macrophages, i.e., their ability to acquire M1 or M2 phenotype depending on the microenvironment signals. M1 exhibit high pro-inflammatory activity and have neurodestructive properties, whereas M2 cells are characterized by low pro-inflammatory activity and pronounced regenerative potential, due to the production of multiple soluble mediators and cytokines, including neurotrophic and immunoregulatory factors, anti-inflammatory substances that provide neuroprotection, stimulate neurogenesis, synaptogenesis, growth and myelinization of axons, thus theoretically substantiating an opportunity of using the potential of M2 macrophages in the treatment of depression. In this work, we studied the effect of soluble factors of human macrophages, polarized into cells with M2 phenotype under the conditions of serum deprivation, upon bone marrow hematopoiesis and peripheral blood cells in a model of stress-induced depression. We have shown enhanced differentiation of hematopoietic stem cells into the granulocyte-macrophage (CFU-GM) lineage, along with increased monocyte population in peripheral blood in the depressive-like murine model. Development of a depressive-like state in the animals was associated with reduced amounts of both erythroid precursors in bone marrow and erythrocytes/hemoglobin in peripheral blood. Intranasal administration of soluble M2 macrophage factors (M2-SFs) for 7 days was accompanied by a corrective effect on the above parameters, being significant for peripheral blood monocytes. The data obtained suggest effectiveness of the M2-SFS anti-inflammatory effects in correcting changes in hematopoiesis caused by social stress in depressive-like animals.

PB1945: CLINICAL AND MOLECULAR CHARACTERISTICS OF CHILDREN WITH DIAMOND-BLACKFAN ANEMIA (DBA): A SINGLE CENTER EXPERIENCE IN ARGENTINA

Background: DBA is an inherited bone marrow failure characterized by erythroblastopenia due to an intrinsic defect in erythropoietic progenitors. Half the patients (pts) present with physical malformations. To date 20 ribosomal proteins (RP) genes associated with DBA and 4 non-RP genes related to non-classical DBA (GATA1, TSR2, EPO, ADA2) have been described. Aims: To describe clinical and molecular characteristics and outcomes of a pediatric cohort with DBA admitted at a single tertiary pediatric center in Argentina Methods: Patients diagnosed with DBA between 2004 and 2020 were retrospectively analyzed. DBA diagnosis was based on constitutional normocytic/macrocytic anemia, reticulocytopenia, paucity of bone marrow erythroid precursors and congenital anomalies. Demographic, clinical, and laboratory data, genetic evaluation and outcomes were recorded. Pts were screened for DNA changes by using a custom designed NGS panel with 19 genes related to DBA (RPS19, RPL11, RPL15, RPL18, RPL27, RPL31, RPL35, RPL35A, RPL5, RPS10, RPS15A, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2 and GATA1). All identified variants were classified according to the American College of Medical Genetics guidelines. Results: Fifteen unrelated pts with DBA were analyzed, 10 (66%) males, median age at diagnosis 3,7months (25days-6yrs). Twelve pts (80%) had physical malformations. All pts showed paucity of bone marrow erythroid precursors, and only two presented with compromise of other linages. Thirteen pts (87%) received treatment with prednisone, 8 (62%) achieved transfusion independence and half of them achieved complete treatment independency, while five pts (33%) did not respond to steroids and are under transfusion regimen. These 5pts developed liver iron overload and are under chelation therapy. No therapy was required in two pts with variants in GATA1 and RPL5. Median follow-up was 7.1years (1.7-16.7 years), no pts received HSCT and no pts died. Molecular variants were detected in 10/15 pts (66%), 8/10 were novel. Eight variants were classified as ¨pathogenic¨ or ¨likely pathogenic” and two as “of uncertain significance”. Three pts (30%) presented variants in RPS19, two (20%) in RPS26, four (40%) in other RP genes and one pt with non-classical DBA, presented a variant in GATA1. Phenotype and molecular results of the pts are summarized in Table1. Image:Summary/Conclusion: In this cohort of pts with DBA, molecular diagnosis was achieved in the majority. RPS19 variants were the most common. Interestingly, all pts with RPS19 variants presented with malformations, one of them had also pancytopenia at onset. As the penetrance of DBA is incomplete, the molecular diagnosis is important for detecting silent phenotype carriers in the family that will allow for an accurate selection of stem cell donors, genetic counseling and tailored treatment.

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.

Poster: 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

Poster: 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

Abstract 1374: The novel PARP1-selective inhibitor AZD5305 has reduced hematological toxicity when compared to PARP1/2 inhibitors in pre-clinical models

Abstract Current clinical poly (ADP-ribose) polymerase (PARP) inhibitors target both PARP1 and PARP2 and they all cause clinical cytopenias with varying severity. Understanding the mechanism underlying the hematological toxicity of these agents is key for the rational design of a best-in-class molecule with greater therapeutic potential, both as monotherapy and in combination with chemotherapy. We validated the rat as a physiologically competent translational model to investigate PARP inhibitor-driven hematological toxicity. Here we demonstrate that in comparison with a representative PARP1/2 inhibitor, olaparib, the novel highly potent PARP1-selective inhibitor and trapper, AZD5305, does not cause hematological toxicity as a monotherapy in pre-clinical rat models at predicted clinically efficacious exposures. Thus, monotherapy toxicity of PARP1/2 inhibitors likely depends on PARP2 inhibition. Next, we proceeded to investigate whether PARP1-selective inhibition would be better tolerated in chemotherapy combinations than PARP1/2 inhibition. We performed a rat in vivo study comparing daily olaparib or daily AZD5305 at matched exposures in combination with one cycle of intravenous carboplatin for 14 days. We show that olaparib and PARP1-selective AZD5305 cause comparable exacerbation of carboplatin-induced peripheral blood effects implicating PARP1 inhibition in combination-driven hematological toxicity. Importantly however, AZD5305+carboplatin showed improved hematological tolerability over olaparib+carboplatin because peripheral reticulocytes and bone marrow erythroid precursor cells recover in the presence of continuous AZD5305 but not in the presence of continuous olaparib. Importantly, this differentiation was maintained in a subsequent rat in vivo study, where daily olaparib or daily AZD5305 were combined with two three-weekly cycles of a higher dose of carboplatin to more closely mimic clinical protocol. AZD5305+carboplatin was associated with a more rapid recovery of reticulocytes, red blood cells and hemoglobin following both cycles of carboplatin. In contrast, olaparib+carboplatin was associated with a slower recovery resulting in a more sustained reduction in red cells and hemoglobin during both the first and second cycle of carboplatin. Thus, in rodents the novel potent PARP1-selective inhibitor AZD5305 has improved hematological tolerability over dual PARP1/2 inhibitors, both as a monotherapy and in carboplatin combinations. Citation Format: Sonja J. Gill, Ruth Macdonald, Carmen Pin, Rob Collins, Emilyanne Leonard, Gareth Maglennon, Andy Pike, Peter Cotton, Glen Hawthorne, Jordan Pugh, Rebecca Sargeant, Daniel Sutton, James Atkinson, Stewart Jones, Sarah Chinery, Mark Anderton. The novel PARP1-selective inhibitor AZD5305 has reduced hematological toxicity when compared to PARP1/2 inhibitors in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1374.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

Diagnosis and management of pure red cell aplasia

Pure red cell aplasia (PRCA) is characterized by normocytic anemia with reticulocytopenia and marked reduction of the bone marrow erythroid precursors. PRCA may be congenital (Diamond-Blackfan anemia) or acquired. Acquired PRCA may present in the context of various backgrounds, the most common type in Japan being idiopathic, thymoma-associated, and large granular lymphocyte leukemia. Idiopathic and secondary PRCA that do not respond to the treatment of the underlying disease are generally treated using immunosuppressive agents. A retrospective study PRCA2004/2006 suggests that maintenance therapy and management of infectious complications is crucial for improving the prognosis in patients with PRCA. Recently, allogeneic stem cell transplantation has been considered as a potential option for the treatment of patients with PRCA who are refractory to immunosuppressive therapy. Sirolimus and roxadustat may be effective for relapsed/refractory PRCA with renal insufficiency and anti-erythropoietin antibody-mediated PRCA, respectively. Some gene mutations were detected in certain patients who had acquired PRCA, and the identification of STAT3 mutations may be useful in PRCA management. A prospective cohort study PRCA2016 has been ongoing in Japan, and novel discoveries provide hope for improving the outcome in patients with PRCA.

Effect of Luspatercept on Biomarkers of Erythropoiesis in Patients (Pts) with Lower-Risk Myelodysplastic Syndromes (LR-MDS) in the Medalist Trial

Effect of Luspatercept on Biomarkers of Erythropoiesis in Patients (Pts) with Lower-Risk Myelodysplastic Syndromes (LR-MDS) in the Medalist Trial

A Novel Choice to Correct Inflammation-Induced Anemia in CKD: Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat.

Anemia is a complication of chronic kidney disease (CKD), primarily due to insufficient secretion of erythropoietin (EPO) by the kidney. Erythropoiesis-stimulating agents (ESAs) are used to treat anemia associated with chronic kidney disease. A poor response to ESAs has been associated with inflammation. Inflammation can affect erythrocytes and its production in many ways, but mainly through the inflammatory cytokine IL-6 to stimulate the synthesis of hepcidin in the liver. Hepcidin causes iron insufficiency, which causes erythrocytes to fail to mature normally. In addition, inhibition of bone marrow erythroid precursor cells by inflammatory cytokines such as IL-1 and TNF-α also affects bone marrow hematopoiesis. These cytokines are also important factors leading to EPO resistance. Roxadustat is a new drug for the treatment of renal anemia. In addition to promoting the production of EPO, clinical trials have shown that it can significantly reduce hepcidin and can potentially be used for the treatment of inflammation-induced anemia in CKD.

Luspatercept Significantly Reduces Red Blood Cell (RBC) Transfusion Burden, Regardless of Gene Mutation Frequency, Spectrum, and Prognostic Significance, Among Patients (Pts) with LR-MDS Enrolled in the MEDALIST Trial

Luspatercept Significantly Reduces Red Blood Cell (RBC) Transfusion Burden, Regardless of Gene Mutation Frequency, Spectrum, and Prognostic Significance, Among Patients (Pts) with LR-MDS Enrolled in the MEDALIST Trial

The impact of spliceosome mutations in MDS.

The impact of spliceosome mutations in MDS.

Issue Highlights - May 2019.

Issue Highlights - May 2019.

Comparative Proteome-Wide Analysis of Bone Marrow Microenvironment of β-Thalassemia/Hemoglobin E.

β-thalassemia/Hb E is a global health issue, which is characterized by a range of clinical symptoms from a mild and asymptomatic anemia to severe disorders that require transfusions from infancy. Pathological mechanisms of the disease involve the excess of unmatched alpha globin and iron overload, leading to ineffective erythropoiesis and ultimately to the premature death of erythroid precursors in bone marrow (BM) and peripheral organs. However, it is unclear as to how BM microenvironment factors contribute to the defective erythropoiesis in β-thalassemia/Hb E patients. Here, we employed mass spectrometry-based comparative proteomics to analyze BM plasma that was collected from six β-thalassemia/Hb E patients and four healthy donors. We identified that the differentially expressed proteins are enriched in secretory or exosome-associated proteins, many of which have putative functions in the oxidative stress response. Using Western blot assay, we confirmed that atypical lipoprotein, Apolipoprotein D (APOD), belonging to the Lipocalin transporter superfamily, was significantly decreased in BM plasma of the tested pediatric β-thalassemia/Hb E patients. Our results highlight that the disease condition of ineffective erythropoiesis and oxidative stress found in BM microenvironment of β-thalassemia/Hb E patients is associated with the impaired expression of APOD protein.

The Hepcidin-Anemia Axis: Pathogenesis of Anemia in Chronic Kidney Disease.

The pathogenesis of anemia in chronic kidney disease (CKD) could be multifactorial. In recent animal studies, hepcidin knockout (KO) mice with adenine-induced CKD did not exhibit anemia and iron deficiency. Hepcidin has emerged as a major player in the development of anemia in CKD. We suspected that erythropoietin (EPO) deficiency may not be the mainstay of anemia in CKD, although relative EPO deficiency could contribute to the failure to increase hemoglobin (Hb) levels. Some factors may interfere with the differentiation of erythroids. Based on previous flow cytometric analysis, the differentiation and maturation of bone marrow erythroid precursors were compared between 2 mouse models of anemia, namely, EPO-KO mice and adenine-induced CKD mice. EPO-KO mice exhibited greater than 50% reduction in the CD71-low/Ter119-high population, which represents a mature erythroid stage in the bone marrow. In contrast, these mice exhibited no reduction in the CD71-high/Ter119-low and CD71-high/Ter119-high cell populations, which represent an early erythroid stage. However, in CKD mice, the percentages of CD71-high/Ter119-low and CD71-high/Ter119-high erythroid cells, which correspond to proerythroblasts and basophilic erythroblasts, respectively, were decreased in bone marrow. Thus, the CKD mice exhibited a decrease in the number of cells expressing transferrin receptor 1 (TfR1) or early stage erythroblasts, which was completely different from the results obtained for EPO-KO mice. Thus, in CKD, decreased expression of TfR1 in erythroblasts as well as increased hepcidin levels in circulation may hamper erythroblast differentiation by decreasing the iron supply, as iron is an indispensable component of erythroblast differentiation. We conclude that deregulated iron metabolism could be the principal cause of anemia in CKD, impeding the differentiation of erythroblasts. We propose that the "hepcidin-anemia axis" is involved in the pathogenesis of CKD-associated anemia. For the treatment of anemia in CKD, declining hepcidin levels are essential for efficient erythropoiesis. Key Messages: These findings have led us to target the hepcidin-anemia axis as a new treatment strategy for anemia in CKD, including via newly developed erythropoiesis-stimulating agent and hypoxia inducible factor stabilizers.

Percentage of Erythroid Cells in Bone Marrow at Diagnosis As a New Prognostic Factor in Patients with Myelodysplastic Syndrome

IntroductionRisk assessment is essential for guiding therapy in patients with myelodysplastic syndromes (MDS). Currently, the most widely used prognostic scoring models are the International Prognostic Scoring System (IPSS) and the revised IPSS (IPSS-R). The prognostic relevance of the percentage of erythroid precursors (EP) in bone marrow (BM) and its relationship with other biological characteristics has been poorly studied, although it has been proposed that a very low percentage of EP in BM may represent a cohort of patients with potentially adverse outcome. Our main aim was to analyze the biological and clinical features of MDS patients according to the percentage of EP in BM at diagnosis and evaluate its prognostic value on survival.Patients and MethodsData from 4,791 de novo MDS patients from the MDS Spanish Registry with available cytogenetics were collected. All patients included were diagnosed based on the 2008 WHO criteria and risk stratification was performed following the IPSS-R. Patients were distributed, according to the percentage of EP in BM, into three groups: less than 15% (EP<15%), between 15% and 49% (EP 15-49%) and equal or more than 50% (EP>49%).Proportions were compared by the Chi-square test. Survival curves were constructed by Kaplan-Meier method and differences between curves were evaluated by log rank tests. Multivariable analysis of survival was performed using Cox's proportional hazards regression model. P-values <0.05 were considered as statistically significant. All statistical analyses were performed by the R software.ResultsThe 4,791 cases were grouped according to the percentage of erythroid cells in BM at diagnosis. In Table 1 are displayed the clinical and biological characteristics of the patients in the whole series and in the three groups based on the EP percentage in BM. The group with EP<15% in BM showed a significantly greater number of cytopenias at diagnosis, a higher number of peripheral blood (PB) and BM blasts, and a higher percentage of patients classified in MDS subtypes with excess blasts. The EP>49% group presented higher rates of patients with refractory anemia with ring sideroblasts (RARS), lower number of PB and BM blasts, and more frequent high-risk cytogenetics. Conversely, the intermediate EP group with EP 15-49% showed less number of cytopenias, higher frequency of good-prognosis cytogenetics, and lower percentage of patients in higher-risk IPSS-R categories.Survival analyses confirmed that age, blast percentage in PB and BM, and IPSS-R cytogenetics are significantly associated with worse survival. A striking difference in survival within groups according to percentage of EP was also found, recognizing EP percentage as an independent risk factor for survival of MDS patients in multivariate analysis. Patients with EP<15% in BM experienced a significantly shorter OS (median, 28 months) in both univariable (P<0.0001) and multivariable analysis (P<0.0001) (Table 2 and Figure 1). The best survival was found for patients with EP 15-49% with a median survival of 58 months. The group with EP>50% in BM had also worse survival than the intermediate group, showing a median survival of 47 months (hazard ratio, 0.79; 95% CI, 0.67 to 0.93; P < 0.005).ConclusionsOur study proves the prognostic impact of the percentage of erythroid precursors in bone marrow in MDS patients, providing evidence that the presence of EP <15% in BM is an independent risk factor for survival. [Display omitted] DisclosuresDíez-Campelo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Analytic characterization of flow cytometric assays for detection of immunoglobulin G on canine erythroid cells, including detection of dog erythrocyte antigen 1 on erythroid precursors.

OBJECTIVE To develop and characterize flow cytometric assays for detecting IgG bound to canine erythrocytes and bone marrow erythroid precursors. SAMPLE Blood samples from 20 healthy and 61 sick dogs with (n = 33) or without (28) immune-mediated hemolytic anemia (IMHA) and bone marrow samples from 14 healthy dogs. PROCEDURES A flow cytometric assay for measurement of IgG on RBCs was developed, and appropriate positive control cells were generated. Analytic and diagnostic performance were characterized. The RBC IgG assay was then combined with density-gradient fractionation of aspirated bone marrow cells and a 2-color process to yield an assay for detecting IgG on nucleated RBCs (nRBCs). Cell sorting and cytologic examination confirmed target cell populations, and anti-dog erythrocyte antigen 1 (DEA1) blood-typing serum was used to generate IgG-positive nRBCs. RESULTS Within- and between-run coefficients of variation for the RBC IgG assay were 0.1% to 13.9%, and > 90% of spiked IgG-positive RBCs were detected. Diagnostic sensitivity and specificity of the assay for detection of IMHA were 88% and 93%, respectively. Cytologic findings for sorted bone marrow fractions rich in early-, mid-, and late-stage nRBCs from 3 healthy dogs indicated 89% to 98% nRBC purity. After IgG coating with anti-DEA1 blood-typing serum, IgG was detected on nRBCs from DEA1-positive, but not DEA1-negative, healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE The developed RBC IgG assay had favorable analytic and diagnostic performance for detection of IMHA in dogs and was successfully adapted to detect IgG on canine nRBCs of various maturation stages. The findings supported the presence of DEA1 on canine nRBCs.

Erythropoietin stimulates fibroblast growth factor 23 (FGF23) in mice and men.

Fibroblast growth factor 23 (FGF23) is a major endocrine regulator of phosphate and 1,25 (OH)2 vitamin D3 metabolism and is mainly produced by osteocytes. Its production is upregulated by a variety of factors including 1,25 (OH)2 vitamin D3, high dietary phosphate intake, and parathyroid hormone (PTH). Recently, iron deficiency and hypoxia have been suggested as additional regulators of FGF23 and a role of erythropoietin (EPO) was shown. However, the regulation of FGF23 by EPO and the impact on phosphate and 1,25(OH)2 vitamin D3 are not completely understood. Here, we demonstrate that acute administration of recombinant human EPO (rhEPO) to healthy humans increases the C-terminal fragment of FGF23 (C-terminal FGF23) but not intact FGF23 (iFGF23). In mice, rhEPO stimulates acutely (24h) C-terminal FGF23 but iFGF23 only after 4days without effects on PTH and plasma phosphate. 1,25 (OH)2 D3 levels and αklotho expression in the kidney decrease after 4days. rhEPO induced FGF23 mRNA in bone marrow but not in bone, with increased staining of FGF23 in CD71+ erythroid precursors in bone marrow. Chronic elevation of EPO in transgenic mice increases iFGF23. Finally, acute injections of recombinant FGF23 reduced renal EPO mRNA expression. Our data demonstrate stimulation of FGF23 levels in mice which impacts mostly on 1,25 (OH)2 vitamin D3 levels and metabolism. In humans, EPO is mostly associated with the C-terminal fragment of FGF23; in mice, EPO has a time-dependent effect on both FGF23 forms. EPO and FGF23 may form a feedback loop controlling and linking erythropoiesis and mineral metabolism.

Effects of Testosterone on Erythropoiesis in a Female Mouse Model of Anemia of Inflammation.

The anemia of inflammation is a common problem in inflammatory and autoimmune diseases. We characterized a mouse model of anemia of chronic inflammation induced by repeated injections of low doses of heat-killed Brucella abortus (HKBA), and determined the effects of T administration on erythropoiesis in this model. Female C57BL/6NCrl mice were injected weekly with HKBA for 10 wk. Weekly injections of T or vehicle oil were started 4 wk later. Control mice were injected with saline and vehicle oil in parallel. HKBA-injected mice had significantly lower hemoglobin, hematocrit, mean corpuscular volume, reticulocyte hemoglobin, transferrin saturation (TSAT), and tissue nonheme iron in liver and spleen, enlarged spleen, and up-regulated hepatic expression of inflammatory markers, serum amyloid A1, and TNFα, but down-regulated IL-6, bone morphogenic protein 6, and hepcidin compared with saline controls. HKBA also reduced serum hepcidin and increased serum erythropoietin. Bone marrow erythroid precursors were substantially reduced in HKBA-injected mice. Cotreatment with T increased the percentage of late-stage erythroid precursors in the bone marrow relative to HKBA-injected and saline controls and reversed HKBA-induced suppression of hemoglobin and hematocrit. T also normalized serum erythropoietin, TSAT, and reticulocyte hemoglobin without correcting the expression of the hepatic inflammation markers. Conclusions are that low-dose HKBA induces moderate anemia characterized by chronic inflammation, decreased iron stores, and suppression of erythroid precursors in the bone marrow. T administration reverses HKBA-induced anemia by stimulating erythropoiesis, which is associated with a shift toward accelerated maturation of erythroid precursors in the bone marrow.

Demographic Characteristics, Survival and Prognostic Factors for Mortality in Cats with Primary Immune-Mediated Hemolytic Anemia.

BackgroundImmune‐mediated hemolytic anemia (IMHA) is uncommon in cats, but may result in severe disease. Demographic predispositions for development of the disease and prognostic factors for mortality have not been investigated previously.Hypothesis/ObjectivesTo explore possible demographic predispositions for development of primary IMHA in cats and to investigate possible prognostic factors for mortality.Animals107 client‐owned cats with IMHA, of which 72 had primary IMHA and 35 had secondary IMHA, and 9,194 control cats.MethodsData were collected retrospectively from records of cats with IMHA, defined by the presence of anemia and concurrent autoagglutination, ghost cells without oxidative damage on fresh blood smear, positive titer in a direct antiglobulin test, or evidence of phagocytosis of erythroid precursors in bone marrow. Odds ratios were calculated to assess the risk of development of primary IMHA in different demographic groups and Cox proportional hazards analysis was conducted to evaluate prognostic factors.ResultsNo sex or breed predisposition was identified for the development of primary IMHA in comparison to the control cats, but cats in the age range 2.1–5.9 years were predisposed. Higher total bilirubin concentration and age were significant negative prognostic factors and higher lymphocyte numbers and serum globulin concentration were positive prognostic factors in a multivariable model.Conclusions and Clinical ImportanceYoung adult cats were more likely to develop primary IMHA than other groups, but no apparent male predisposition was identified in this study, contrary to previous reports. Several prognostic factors were identified, which may be helpful in guiding clinical practice in the future.