Number Of Erythroid Progenitor Cells Research Articles

CD45- erythroid progenitor cells promote lymph node metastasis in gastric cancer by inducing a hybrid epithelial/mesenchymal state in lymphatic endothelial cells.

Specific mechanisms of lymph node (LN) metastasis in early-stage gastric cancer (GC) have not been elucidated. The role of anemia, a vital clinical feature of GC, in LN metastasis is also unclear. Since the number of erythroid progenitor cells (EPCs) is increased in chronic anemia, we investigated its association with LN metastasis in GC. Flow cytometry and immunofluorescence analyses were performed to sort and study EPCs from the circulation and tumors of patients with stage I-III GC. The effect of these EPCs on the activation of T and B cells and on the functions of lymphatic endothelial cells (LECs) was determined, and their ability to promote LN metastasis was evaluated using a footpad-popliteal LN metastasis model based on two human adenocarcinoma GC cell lines in nude mice. The prognostic value of EPCs was also analyzed. The proportion of CD45- EPCs was higher in the mononuclear cells in the circulation, tumors, and LNs of GC patients with LN metastasis (N+) than in those of GC patients without LN metastasis (N0). In N+ patients, CD45- EPCs were more abundant in metastatic LNs than in non-metastatic LNs. Lymphatic vessel endothelial hyaluronan receptor 1 immunoreactivity in tumors revealed that CD45- EPCs were positively associated with nodal stages and lymph vessel density. Furthermore, CD45- EPCs increased LEC proliferation and migration through their S100A8/A9 heterodimer-induced hybrid epithelial/mesenchymal (E/M) state; however, they did not influence the invasion and tubulogenesis of LECs or T and B cell proliferation. CD45- EPCs promoted LN metastasis in vivo; the S100A8/A9 heterodimer mimicked this phenomenon. Finally, CD45- EPCs predicted the overall and disease-free survival of stage I-III GC patients after radical resection. The CD45- EPCs accumulated in GC tissues and metastatic LNs and promoted LN metastasis via the S100A8/9-induced hybrid E/M state of LECs, which was the specific mechanism of LN metastasis in the early stages of GC.

Macrophages Provide Essential Support for Erythropoiesis, and Extracellular ATP Contributes to a Erythropoiesis-Supportive Microenvironment during Repeated Psychological Stress.

Psychological stress is a significant contributor to various chronic diseases and affects multiple physiological processes including erythropoiesis. This study aimed to examine the tissue-specific contributions of macrophages and extracellular ATP, as a signal of disturbed tissue homeostasis, to erythropoiesis under conditions of repeated psychological stress. Adult male BALB/c mice were subjected to 2 h daily restraint stress for seven consecutive days. Clodronate-liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to the first restraint procedure, as well as newly recruited macrophages, every third day for the duration of the experiment. Repeated stress induced a considerable increase in the number of erythroid progenitor cells as well as in the percentage of CD71+/Ter119+ and CD71-/Ter119+ cells in the bone marrow and spleen. Macrophage depletion completely abolished the stimulative effect of repeated stress on immature erythroid cells, and prevented stress-induced increases in ATP levels, P2X7 receptor (P2X7R) expression, and ectonucleotidase CD39 activity and expression in the bone marrow and spleen. The obtained results demonstrate the stimulative effects of repeated stress on erythroid cells, extracellular ATP levels, P2X7R expression, CD39 activity and expression within the bone marrow and spleen, as well as the essential role of macrophages in stress-induced changes.

Identification and Analysis of Mouse Erythroid Progenitor Cells.

The most common cell type in the human body, the red blood cell or erythrocyte, has a life span of approximately 3months. To compensate for this massive cellular requirement and short life span, the major blood producing tissues contain vast numbers of erythroid progenitor cells. Erythroid progenitors differentiate progressively from hematopoietic stem cells to committed erythroid progenitors to reticulocytes lacking a nucleus and finally to functionally mature erythrocytes in the circulation. Different erythroid progenitor activity, representative of distinct stages of erythropoiesis, can be observed using semisolid colony assays. Distinct stages of erythroid maturation can also be monitored by flow cytometry. Here, we discuss the range of different technical approaches that are used to identify and quantify erythroid progenitors, with particular focus on the mouse as a model system.

Potential Therapeutic Applications of Jak2 Inhibitors and Hif2a-ASO for the Treatment of β-Thalassemia Intermedia and Major

β-Thalassemia is one of the most common genetic red blood cell (RBC) disorders characterized by reduced (such as in non-transfusion dependent thalassemia or NTDT) or absent (such as in transfusion dependent thalassemia or TDT) production of β-globin chains. Ineffective erythropoiesis (IE) with consequent anemia leads to extra-medullary hematopoiesis (EMH), splenomegaly and iron overload mediated by low levels of hepcidin. We previously demonstrated that IE in β-thalassemia is associated with increased proliferation and reduced differentiation of erythroid progenitors (Libani et al, 2008, Blood). This is mediated by increased production of erythropoietin (EPO), which activates the downstream JAK2 kinase in erythroid progenitors. As a consequence, hepatosplenomegaly may result, often requiring splenectomy to prevent serious morbidities and mortality. The increased synthesis of EPO in thalassemia is the result of hypoxia. Hypoxia Inducible Factor-2a (HIF2a) is a central mediator of cellular adaptation to hypoxia and stimulates renal and hepatic EPO synthesis. Furthermore, splenomegaly in thalassemia also exacerbates the anemia, as a large proportion of the circulating RBC are engulfed and eliminated by an enlarged spleen.Therefore, we hypothesized that targeting the EPO/JAK2 pathway limits the number of erythroid progenitor cells and reduces the splenomegaly, and it could be utilized as an alternative to splenectomy. We utilized JAK2 inhibitors or HIF2a antisense oligonucleotides (HIF2a-ASO) to target the EPO/JAK2 pathway. Moreover, we hypothesized that combination of these drugs with blood transfusion therapy will further reduce the splenomegaly and, eventually, improve the blood transfusion regimen.We tested two commercially available JAK2 inhibitors in mice affected by NTDT (Hbbth3/+): INCB018424 (Ruxolitinib) and TG101348 (Fedratinib, SAR302503). Both drugs were administered for 10 days, twice daily by oral gavage at a dose of 180 and 120 mg/Kg respectively. A mild reduction in hemoglobin (Hb) levels, (in the range of 9%), was observed in animals treated with both inhibitors when compared to vehicle- treated mice. Splenomegaly was significantly reduced with both inhibitors (up to 56% in reduction). Flow cytometry studies on spleen cells revealed that animals receiving the inhibitors exhibited a reduction in the number of erythroid progenitors compared to the placebo-treated animals . In parallel, we performed pharmacokinetic[BM1] studies using Hif2a-ASO. Animals received Hif2a-ASO at a dose of 25 mg/kg twice weekly for 10 days or 3 weeks by IP injections. After 10 days of treatment the spleen weight was reduced 58%, while the Hb level was reduced in the range of 27%%. After 3 weeks the effect observed on the anemia was more pronounced, with a reduction of more that 40% in Hb levels, while the spleen reduction was 36%. We then compared and combined these drugs with blood transfusion using Hbbth3/+ mice. Blood transfusion reduced splenomegaly 49% and 53% when compared, respectively, to non-transfused controls and animals treated with JAK2 inhibitors alone. When transfusion was combined with the administration of JAK2 inhibitors for 10 days, the spleen size was further reduced (72% when compared with non-transfused controls). Combination of Hif2a-ASO and blood transfusion is in progress. We then tested the JAK2 inhibitors in mice affected by TDT. TDT animals are generated by engrafting WT mice with Hbbth3/th3 fetal liver cells. Following engraftment, these mice show a large splenomegaly and rapidly become dependent on chronic blood transfusion for survival (Gardenghi et al, 2007, Blood). Preliminary studies suggest that administration of JAK2 inhibitors for 10 days, together with blood transfusion, further reduces spleen weight by 71% compared to transfusion alone. Combination of Hif2a-ASO and blood transfusion in these animals is also in progress. In summary, JAK2 inhibitors and Hif2a-ASO reduce splenomegaly by targeting the EPO/JAK2 pathway and limiting the excessive proliferation of erythroid cells. Therefore, these drugs could be effective in reversing the splenomegaly and may offer an important approach to splenectomy. Additional studies are in progress to evaluate the outcome of combination therapy on the efficacy of transfusions.[BM1]These aren't pharmacokinetic studies. Suggest just striking the word DisclosuresCasu:Medgenics LLC: Research Funding; Ionis Pharmaceuticals: Research Funding; Merganser Biotech: Research Funding. Aghajan:Ionis Pharmaceuticals: Employment, Equity Ownership. Guo:Ionis Pharmaceuticals: Employment, Equity Ownership. Melchiori:Adaptimmune Ltd: Employment. Ramos:Novartis: Employment.

Essential role of Ufm1 conjugation in the hematopoietic system.

Protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins plays a pivotal role in a wide range of cellular functions and signaling pathways. The Ufm1 conjugation system is a novel ubiquitin-like system that consists of Ufm1, Uba5 (E1), Ufc1 (E2), and less defined E3 ligase(s) and targets. Despite its discovery more than a decade ago, its biological functions and working mechanism remains poorly understood. Recent genetic studies using knockout mouse models provide unambiguous evidence for the indispensable role of the Ufm1 system in animal development and hematopoiesis, especially erythroid development. In this short review, we summarize the recent progress on this important protein modification system and highlight potential challenges ahead. Further elucidation of the function and working mechanism of the ufmylation pathway would provide insight into disease pathogenesis and novel therapeutic targets for blood-related diseases such as anemia.

Evaluation of median survival patients with acute lymphoblastic leukemia exposed to ionizing radiation by the Chornobyl accident, depending on the aminoacid composition of stromal fibroblasts bone marrow and indicators of myelogram

To investigate the aminoacid composition in the stromal fibroblasts of bone marrow and indexes of hemogram in children with acute lymphoblastic leukemias, which exposed to radiation from the Chornobyl accident, for evaluation of the median survival. In the study 54 patients with acute lymphoblastic leukemia, residents of Kyiv, Zhytomyr and Chernihiv regions of Ukraine were involved. Effectiveness of stromal fibroblasts colony forming units of bone marrow and aminoacid composition of fibroblasts were studied. Bone marrow examination was conducted on day33 and after stop of chemotherapy, the median survival of patients and exposure dose were evaluated. In children with acute leukemias effectiveness of stromal fibroblasts of bone marrow was 2-2,3 times lower than normative. Correlation (rs = +0,83) between the number of erythroid progenitor cells in the bone marrow and proline content in stromal fibroblasts established . The correlation between patients median survival and number of granulocytes in myelogram (rs = + 0,82) was proven. Mean exposure doses in patients was (5,86 ± 1,11) mSv and it did not correlate with myelogram results, effectiveness of stromal fibroblasts of bone marrow and median survival. The median survival of patients with acute leukemias depends on the content of amino acids in stro mal fibroblasts of bone marrow and recover of hematopoiesis after chemotherapy.

Chromatin Modifying Agents Promote the Ex Vivo Production of Functional Human Erythroid Progenitor Cells

AbstractAbstract 340Presently, blood transfusion products (TP) are composed of terminally differentiated cells with a finite life span. We attempted to develop an alternative TP which would be capable of generating additional red blood cells (RBC). Several histone deacetylase inhibitors (HDACIs) were used in vitro to reprogram cord blood (CB) CD34+ cells to differentiate to erythroid progenitor cells (EPC). We demonstrated that CB CD34+ cells in the presence of HDACIs (SAHA, VPA and TSA), and a combination of cytokines SCF, IL-3, TPO and FLT3, promoted expansion of CD34+ cells and CD34+CD90+ cells as compared to cultures containing cytokines alone. Addition of VPA resulted in the greatest expansion of CD34+ cells, CD34+CD90+cells+ (59.4 fold, p=0.01; 66.7 fold, p=0.02, respectively) as compared to SAHA and TSA. VPA also led to the generation of the greatest absolute number of EPC cells (14.9×106, p=0.002), approximately a 5500 fold in the numbers of assayable EPC, as compared to primary CB.The single cell analyses of CB CD34+ cells (Day0) and single CD34+ reisolated from ex-vivo cultures pretreated with cytokines alone or cytokines+VPA demonstrated an skewed differentiation program of CD34+ cells to EPC (>94%, p=0.003) compared to CB CD34+(50%) and cytokines alone (29%).We investigated the expression of lineage specific phenotypic markers expressed by CD34+ cells exposed to cytokines alone or cytokines plus VPA. The FACS analyses showed a significantly greater proportion of CD34+CD36+ (52.4% vs 21.0%) CD36+CD71+(44.5% vs7.6%), CD36+GPA+(12.8% Vs 4.0%) and CD71+GPA+(22.2% vs 6.3%) cells with lower numbers of CD19+(2.8% vs 13.6%) cells, CD14+(2.0% vs 8.9%), CD15+(1.8 vs 6.9%) in VPA treated CD34+ cells as compared to cytokines alone.We monitored the relative expression of a group of genes characteristic of both primitive HPC and erythroid commitment (Bmi1, Dnmt1, Ezh2, Smad5, Eklf, GATA1, GATA2, EpoR and Pu.1). Q-PCR was performed on CD34+cells reisolated from cultures treated with cytokines alone or cytokines plus VPA and compared to primary CB CD34+ cells. The expression of genes associated with retention of the biological properties of the primitive HPC (Bmi1-2.6 fold, Dnmt1-10.3 fold and Ezh2-4.8 fold) and erythroid lineage specific genes (Smad5-6.2 fold, GATA2-3.7 fold) were upregulated and Pu.1 (0.6-fold), GATA1(1.9 fold) were downregulated as compared to cytokines alone. However, expression of EpoR and Eklf were similar in the two cell populationsHistone acetylation study showed that the CB CD34+ cells and VPA treated CD34+ cells had a significant proportion of acetylated H3K9 cells, 52.2% and 56.1% respectively, while this population was virtually absent in CD34+ cells exposed to cytokines alone (1.3%, p=0.001). ChIP assay demonstrated a varying degree of H3K9/14 and H3K27 acetylation within the promoters of VPA treated CD34+ cells for GATA2 (7.4 fold, 7.2 fold), Eklf (7.4 fold, 9.7 fold), Pu.1(4.5fold, 4.8 fold), EpoR (2.3 fold, 4.7 fold) and GATA1(4.7 fold, 2.9 fold). The acetylation of cytokines treated CD34+ cells were much lower than VPA treated CD34+ cells.The VPA treated cell product after 9 days (supplemented with SCF, Epo and IL-3 for 2 additional days) compared to 7 days contained a greater percentage of EPC and erythroid precursor cells CD34+CD36+(24.9% vs 23.0%), CD36+GPA+(33.9% vs 18.8%), CD36+. CD71+(55.8% vs 37.8%), CD71+GPA+(33.9% vs 20.5%) and CD34+CXCR4+(28.8% vs 21.0 %). The TP contained very limited number of CD19+(1.4%), CD14+(11.11%) or CD15+(6.8%) of cells. Approximately 50 % of the cells present in the TP expressed the chemokine receptor CXCR4.We next evaluated the behavior of ex vivo expanded cell product following transfusion into sublethally irradiated NOD/SCID mice. FACS analyses of mice peripheral blood (PB) on serial days showed evidence of circulating nucleated erythroid and enucleated red cells. The greatest number of circulating human RBC (12.4%±6.8%) was observed on day5. RT-PCR analyses on the PB of mice on day 15 revealed the presence of erythroid cells containing both human adult and fetal hemoglobin. On day 15 the mice were sacrificed and the degree of human cells engraftment in the marrow were predominately hu -CD45+ (7.4%), CD34-CD36+(1.8%), CD36 (4.5%) and GPA+(1.7%) with no evidence of CD33+, CD14+, CD19+ and CD41+ cells. The ex vivo generated EPC-TP likely represents a paradigm shift in transfusion medicine due to its continued ability to generate additional RBC. Disclosures:No relevant conflicts of interest to declare.

Effect of Neopterin on Splenic Erythropoiesis in Mice

Abstract Neopterin is produced by monocytes and is a biomarker for inflammation. We recently found that neopterin induces stromal cells to produce cytokines that suppress erythropoiesis in the bone marrow of mice. Despite this suppression, the peripheral hematocrit values are not decreased. The goal of the present study was to determine whether the maintenance of hematocrit values is caused by a neopterin-induced acceleration of extramedullary erythropoiesis. We examined the effects of intravenously injected neopterin on splenic erythropoiesis in C57BL/6J mice and found a marked increase (259% of the pretreatment level) in the number of splenic erythroid progenitor cells (BFU-E and CFU-E) after a slight temporal decrease in the number of erythroid progenitor cells. These results suggest that neopterin increases splenic erythropoiesis to compensate for the suppression of erythropoiesis in the bone marrow, resulting in the maintenance of hematocrit levels in peripheral blood.

Inflammatory biomarker, neopterin, predominantly enhances myelopoiesis, which suppresses erythropoiesis via activated stromal cells

Neopterin is produced by monocytes and is a useful biomarker for inflammation. We found previously that neopterin enhanced myelopoiesis but suppressed B-lymphopoiesis triggered by the positive and negative regulations of cytokines produced by stromal cells in mice. The effects of neopterin on erythropoiesis during the enhancement of myelopoiesis were determined in the present study using C57BL/6J mice. The intravenous injection of neopterin into mice resulted in a prolonged decrease in the number of femoral erythroid progenitor cells (BFU-Es and CFU-Es), whereas the number of femoral myeloid progenitor cells (CFU-GMs) was increased. Interestingly, the oscillatory changes in the number of erythroid progenitor cells were reciprocal to those of myeloid progenitor cells. The expression of Cdc42, a regulator of the balance between erythropoiesis and myelopoiesis, was down-regulated, implying that the suppression of erythropoiesis is due to myelopoietic predominance. Furthermore, the expression of SDF-1 in stromal cells, a negative regulator of erythropoiesis, was up-regulated. These results suggest that neopterin facilitates myelopoiesis in the bone marrow by suppressing erythropoiesis, thereby contributing to the potential up-regulation of inflammatory process.

Ex Vivo-Generated CD36 + Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36(+) EPCs expanded and differentiated from CD34(+) HSCs and assessed the CD36(+) EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36(+) EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36(+) EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36(+) EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36(+) EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100- and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

Subcutaneous administration of genistein prior to lethal irradiation supports multilineage, hematopoietic progenitor cell recovery and survival

Purpose: Genistein, a non-toxic isoflavone from soybeans, has immunomodulating and radioprotective properties. In this study we investigated the mechanism for genistein-induced radioprotection by evaluating the recovery of bone marrow cells and peripheral blood hematology in lethally irradiated mice.Materials and methods: CD2F1 male mice received a single subcutaneous injection of genistein (200 mg/kg) 24 h prior to a lethal, total body irradiation dose (8.75 Gy) of cobalt-60 gamma radiation. Survival and hematopoietic reconstitution were evaluated over nine weeks post-irradiation. Hematopoietic progenitor colony-forming cell assays were used to assess the reconstitution of bone marrow after radiation-induced myelosuppression.Results: A total of 97% of genistein-treated mice survived after 30 days while 31% of vehicle-treated and 0% of untreated mice survived. The improvement in survival was related to accelerated neutrophil and platelet recovery, resulting from earlier and more pronounced multilineage, hematopoietic progenitor cell reconstitution in the femoral marrow compartment. Myeloid and erythroid progenitor cell numbers at day 15 post-irradiation were 6-fold to 20-fold higher in genistein-treated animals than in control animals.Conclusions: These results demonstrate that a single subcutaneous administration of genistein 24 h before irradiation provides significant radioprotection to the hematopoietic progenitor cell compartment.

UNRESOLVED ISSUES IN DIALYSIS: Managing Erythropoietin Hyporesponsiveness

ABSTRACTThe anemia of chronic kidney disease is associated with cardiovascular disease, decreased quality of life, and mortality. The introduction of recombinant human erythropoietin (rHuEPO) has transformed the management of this condition. However, a significant proportion of patients fail to respond to even high doses of rHuEPO. Several factors have been implicated in the hyporesponsiveness to rHuEPO. Iron deficiency, whether absolute or functional, is considered the most important, and maintenance of adequate iron stores reduces rHuEPO requirements among patients on hemodialysis. However, traditional indices of iron that are currently utilized may not reflect iron stores accurately, and there is also increasing concern regarding the potential long‐term toxicity of parenteral iron therapy. Infection and inflammation also influence the response to rHuEPO, both by disruption of iron metabolism and by eliciting the release of cytokines that inhibit erythropoiesis. Oxidative stress may contribute to rHuEPO hyporesponsiveness directly by promoting lipid peroxidation in cell membranes, leading to increased erythrocyte fragility and reduced life span and also through its strong association with inflammation. Severe hyperparathyroidism can lead to a reduced number of erythroid progenitor cells. Inadequate dialysis dose, aluminum overload, nutritional factors such as deficiencies of carnitine, vitamin B12, folic acid, and vitamin C can also reduce the efficacy of rHuEPO therapy. Hyporesponsiveness to rHuEPO presents a challenge to both diagnosis and management in an era where optimizing response to rHuEPO is critical both in limiting the burgeoning costs of anemia management and improving clinical outcomes in the dialysis population.

Appearance of central nervous system lupus during corticosteroid therapy and warfarinization in a patient with pure red cell aplasia and antiphospholipid syndrome

A 48-year-old woman presented to our hospital with epigastralgia and erythema on the left dorsalis pedis. Her medical history included deep venous thrombosis three months prior to admission to our hospital. Upon admission it was determined that she had severe anemia (hemoglobin level 4.6 g/dl). Bone marrow analysis indicated a markedly decreased number of erythroid progenitor cells. A skin biopsy specimen of the erythema revealed microthrombus. Anticardiolipin-beta2GPI antibody and lupus anticoagulant were positive. The patient was diagnosed with pure red cell aplasia (PRCA) and antiphospholipid syndrome (APS). After steroid pulse therapy and warfarinization, her anemia and purpura improved. Three months later she developed depression with positive anti-ribosomal P protein antibody that was indicative of central nervous system lupus. Although her psychometric condition did not respond to steroid pulse therapy, improvement was seen after she received three courses of cyclophosphamide pulse therapy. We report a rare case of CNS lupus that developed during corticosteroid therapy and warfarinization in a patient with PRCA and APS.

Effect of spaceflight on human stem cell hematopoiesis: suppression of erythropoiesis and myelopoiesis

Humans subjected to periods of microgravity develop anemia, thrombocytopenia, and abnormalities in red blood cell structure. The causes of these abnormalities are complex and unclear. The in vitro effects of spaceflight on hematopoietic cell proliferation and differentiation were investigated during the space shuttle missions STS-63 (Discovery) and STS-69 (Endeavour). CD34+ bone marrow progenitor cells were cultured in liquid suspension culture and on hematopoietic supportive stromal cells using hollow-fiber culture modules. One set of cultures was maintained at microgravity (flight cultures) for the last 8-10 days of culture and a second control was at full gravity (ground control). Over the 11- to 13-test-day period, ground control culture total cell number increased 41.0- to 65.5-fold but flight culture total cell number increased only 10.1- to 17.6-fold (57-84% decrease). Comparing ground control cultures and microgravity cultures, respectively, for progenitor cell content, myeloid progenitor cell numbers expanded 2.6- to 17.5-fold compared with 0.9- to 7.0-fold and erythroid progenitor cell numbers expanded 2.0- to 4.1-fold in ground control cultures but actually declined at microgravity (>83% reduction). Moreover, microgravity cultures demonstrated accelerated maturation/differentiation toward the macrophage lineage. These data indicate that spaceflight has a direct effect on hematopoietic progenitor cell proliferation and differentiation and that specific aspects of in vitro hematopoiesis, particularly erythropoiesis, involve gravity-sensitive components.

In vivo effects of interleukin-11 and stem cell factor in combination with erythropoietin in the regulation of erythropoiesis.

In this study we evaluated the in vivo effects of interleukin-11 (IL-11) and stem cell factor (SCF), in combination with erythropoietin (EPO) on murine erythropoiesis. Mice were treated for 7 d with IL-11. SCF and EPO, each at three dose levels. In total, 27 different dose combinations were tested. IL-11 as well as SCF could only marginally stimulate erythroid progenitor cell numbers, but IL-11 in combination with SCF was able to increase BFU-E and CFU-E numbers 4-fold, in the absence of exogenous EPO. This resulted in an increased reticulocyte count. In contrast with the stimulatory effect on immature erythroid cell stages, IL-11 treatment induced a mild anaemia, which probably resulted from a plasma volume expansion. The additional treatment with EPO resulted in strong synergistic effects on CFU-E numbers. The combination of high-dose IL-11 and high-dose SCF was able to increase the overall efficiency of EPO-induced erythroid amplification, which was reflected by a left-shift of the in vivo EPO dose-response curve. The stimulating effects of IL-11 and SCF were further demonstrated when the effects on the reticulocyte count of a single high-dose EPO injection were assessed in normal and SCF+IL-11 treated mice. Whereas a single EPO dose increased the reticulocyte count by a factor of 3, IL-11 + SCF pretreatment increased this to a factor of 7. This study shows that in vivo SCF and IL-11 are important modulators of red blood cell production. First, these factors probably increase the input from the stem cell compartment into the erythroid lineage, where subsequently EPO is required for further amplification. Additionally, however, IL-11 and SCF increase the overall efficiency of EPO-induced amplification, probably due to a stimulatory effect on late-stage erythroid cells and to a redistribution of cells from marrow to spleen.

Ligand-Dependent Repression of the Erythroid Transcription Factor GATA-1 by the Estrogen Receptor

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

Apoptosis mediates and thymidine prevents erythroblast destruction in folate deficiency anemia.

Deficiency of the vitamin folic acid causes pancytopenia by decreasing the production of new blood cells. Although impaired DNA synthesis and destruction of hematopoietic cells have been implicated, the mechanism by which folate deficiency decreases blood cell production is uncertain. An in vitro model of folate-deficient erythropoiesis was developed by using proerythroblasts isolated from folate-deficient mice that were infected with Friend leukemia virus. Proerythroblasts from folate-deficient mice had one-tenth the total folate as did proerythroblasts from control mice. The folate-deficient proerythroblasts underwent apoptosis, a form of programmed cell death, after 20-32 h in culture in folate-deficient medium. At the time of apoptosis the cells had differentiated into the later erythroblast stages and some had begun hemoglobin synthesis. Addition of either folic acid or thymidine, but not deoxycytidine or inosine, to the folate-deficient medium prevented the apoptosis and permitted proliferation and differentiation of the proerythroblasts into reticulocytes. The prevention of apoptosis by thymidine indicates (i) that decreased thymidylate synthesis plays a role in erythroblast apoptosis and the anemia of folate deficiency and (ii) that DNA cleavage is likely to be a primary event in the apoptosis of folate-deficient erythroblasts. Apoptosis of erythroblasts in the late stages of differentiation leads to decreased erythrocyte production and to anemia. The increased erythropoietin produced in response to the anemia increases the number of erythroid progenitor cells in the differentiation stages preceding those in which the cells undergo apoptosis. This population shift to earlier stage erythroblasts and proerythroblasts is characteristic of bone marrows of individuals with folate deficiency anemia.

Erythropoietin in cardiac surgery.

Erythropoietin is the primary growth factor for red blood cells. A glycoprotein hormone synthesized by the kidneys, erythropoietin serves to increase red blood cell production in response to tissue hypoxia. It exerts its effect by increasing the numbers of erythroid progenitor cells in the bone marrow, and by increasing the rate at which their development is accomplished. With the introduction of recombinant erythropoietin in 1987, an important pharmacological agent became available for the manipulation of erythropoiesis. While used primarily for the treatment of the anemia of renal failure, recombinant erythropoietin has also shown usefulness in treating other types of anemias in which the endogenous erythropoietin response is insufficient. Perioperative use of the drug grew as a natural extension of this, and erythropoietin has been applied to correct preoperative anemia, augment autologous blood donation, and improve postoperative red cell recovery. Analysis of these perioperative clinical studies reveals success in these areas, but it also reveals that closer attention to the physiology of the natural response, and to the pharmacology of the recombinant product, might significantly improve results. Such an improvement in efficacy is both desirable and necessary when use of the drug is viewed in the setting of today's changing health care environment. By optimizing dosing schedules and targeting the drug to those most at risk for red cell transfusion, recombinant erythropoietin will likely become an important tool in efforts to achieve the elusive goal of bloodless cardiac surgery.

Productive infection of in vitro generated haemopoietic progenitor cells from normal human adult peripheral blood with parvovirus B19: studies by morphology, immunocytochemistry, flow-cytometry and DNA-hybridization.

Parvovirus B19 exerts a highly selective cytopathic effect on erythroid progenitor cells. Studies so far on the pathogenesis of B19-infection have been performed using bone marrow samples providing large amounts of erythroid progenitor cells. Extensive study, however, has been hampered by the limited access to bone marrow samples. We have designed a liquid culture method allowing the generation of large numbers of erythroid progenitor cells, initiating cultures with CD3- and CD14-poor peripheral blood mononuclear cells. Following a 12 d preincubation in liquid cultures containing recombinant human interleukin 3 (rhIl-3) and recombinant human erythropoietin (rhEpo), cells harvested from the liquid cultures were exposed to B19-containing plasma, followed by a further cultivation in liquid culture for up to 96 h. Cells expressing the CD13 and the glycophorin A (GlyA) antigens, respectively, were monitored sequentially by flow-cytometry, demonstrating a selective inhibition of GlyA-positive cells following B19-inoculation. Typical morphological changes were observed on cytocentrifuge-spots, and typical giant-cells were identified as staining for GlyA. Productive infection by B19 was demonstrable, as B19-DNA increased by about x 100 after 72 h of culture. The liquid culture method generating erythroid target cells for effective infection by B19 virus promises to be a useful and easily accessible tool for further research on B19 infection of haemopoietic cells.

Effect of subcutaneously administered human recombinant erythropoietin on erythropoiesis in patients with myelodysplasia

In a phase II study, 12 patients with a myelodysplastic syndrome (MDS) and anaemia (nine transfusion-dependent) were treated with recombinant human erythropoietin (rHuEpo) to assess the therapeutic effect on erythropoiesis and on transfusion requirement. Patients with a low risk of developing acute leukaemia were included, i.e. refractory anaemia (RA), RA with ringed sideroblasts (RARS) and RA with excess blasts (RAEB), providing the percentage of myeloblasts in the bone marrow did not exceed 10%. Recombinant HuEpo treatment was initiated at a dose of 50 units/kg body weight and administered subcutaneously three times weekly. At 3-week intervals the dose was increased with 50 units/kg per injection, until after 15 weeks a maximum dose of 250 units/kg three times weekly was reached. All patients completed the study. Recombinant HuEpo was well tolerated and no serious side effects were seen. There was no evidence of the emergence of a new malignant clone in response to rHuEpo as shown by sequential karyotyping. In none of the patients was an increase in haemoglobin level or a diminished red blood cell transfusion requirement seen. In four out of 10 evaluable sequential bone marrow smears, an increase in erythropoiesis was seen, suggesting stimulation of ineffective red cell production. One of these patients also showed a rise in reticulocyte count. The number of erythroid progenitor cells (BFU-E and CFU-E) in blood and bone marrow was not affected by rHuEpo treatment. Also no change in the number of myeloid progenitor cells (CFU-GM) in blood and bone marrow was noted. In conclusion, subcutaneous treatment with rHuEpo at dosages up to 250 units/kg body weight (three times weekly) fails to increase the haemoglobin level or to diminish the transfusion requirement in patients with MDS and anaemia. It is unclear whether higher doses of rHuEpo are effective or whether patients with less severe anaemia who are transfusion independent, have a higher likelihood of response.