Bone Marrow Erythroid Progenitor Cells Research Articles

Expression of microRNA-155 in thalassemic erythropoiesis.

Ineffective erythropoiesis (IE) is the primary cause of anemia and associated pathologies in β-thalassemia. The characterization of IE is imbalance of erythroid proliferation and differentiation, resulting in increased erythroblast proliferation that fails to differentiate and gives rise to enucleate RBCs. MicroRNAs (miRs) are known to play important roles in hematopoiesis. miR-155 is a multifunctional molecule involved in both normal and pathological hematopoiesis, and its upregulation is observed in patients with β-thalassemia/HbE. However, the expression and function of miR-155, especially in β-thalassemia, have not yet been explored. To study miR-155 expression in thalassemia, erythroblast subpopulations, CD45-CD71+Ter-119+ and CD45-CD71-Ter-119+ were collected from β IVSII-654 thalassemic bone marrow. Additionally, a two-phase culture of mouse bone marrow erythroid progenitor cells was performed. Expression of miR-155 and predicted mRNA target genes, c-myc, bach-1 and pu-1, were determined by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and normalized to small nucleolar RNA (snoRNA) 202 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. To investigate the effect of miR-155 expression, erythroblasts were transfected with miR-inhibitor and -mimic in order to elevate and eliminate miR-155 expression, respectively. Erythroid cell differentiation was evaluated by Wright-Giemsa staining and flow cytometry. miR-155 was upregulated, both in vivo and in vitro, during erythropoiesis in β-thalassemic mice. Our study revealed that gain- and loss of function of miR-155 were involved in erythroid proliferation and differentiation, and augmented proliferation and differentiation of thalassemic mouse erythroblasts may be associated with miR-155 upregulation. miR-155 upregulation in β-thalassemic mice significantly increased the percentage of basophilic and polychromatic erythroblasts. Conversely, a significant decrease in percentage of basophilic and polychromatic erythroblasts was observed in β-thalassemic mice transfected with anti-miR-155 inhibitor. We also examined the mRNA targets (c-myc, bach-1 and pu-1) of miR-155, which indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation. miR-155 regulates IE in β-thalassemia via c-myc expression controlling erythroblast proliferation and differentiation.

Non-Permissive Parvovirus B19 Infection: A Reservoir and Questionable Safety Concern in Mesenchymal Stem Cells.

Mesenchymal stromal/stem cells (MSCs) are multipotent cells with differentiation, immunoregulatory and regenerative properties. Because of these features, they represent an attractive tool for regenerative medicine and cell-based therapy. However, MSCs may act as a reservoir of persistent viruses increasing the risk of failure of MSCs-based therapies and of viral transmission, especially in immunocompromised patients. Parvovirus B19V (B19V) is a common human pathogen that infects bone marrow erythroid progenitor cells, leading to transient or persistent anemia. Characteristics of B19V include the ability to cross the placenta, infecting the fetus, and to persist in several tissues. We thus isolated MSCs from bone marrow (BM-MSCs) and fetal membrane (FM-MSCs) to investigate their permissiveness to B19V infection. The results suggest that both BM- and FM- MSCs can be infected by B19V and, while not able to support viral replication, allow persistence over time in the infected cultures. Future studies are needed to understand the potential role of MSCs in B19V transmission and the conditions that can favor a potential reactivation of the virus.

Upregulation of Pro-inflammatory Cytokine Genes by Parvovirus B19 in Human Bone Marrow Mesenchymal Stem Cells.

Chronic inflammation plays a prominent role in cancer initiation and development. On the other hand, the Inflammation can be established by a number of factors such as viral infections. Parvovirus B19 (B19V) is a pathogen with widespread infection, which infects bone marrow erythroid progenitor cells. It has been shown that B19V can also enter human bone marrow mesenchymal stem cells (BM-MSCs). In this study, we hypothesized that BM-MSCs as the main cellular component of bone marrow niche may be induced to secret pro-inflammatory cytokines after B19V infection. BM-MSCs were cultured up to passage 3. The cells were then subjected to nucleofection to transfer a plasmid containing B19V genome. After 36h, total RNA was extracted and the expression levels of IL-1β, IL-6, TNF-α and NF-κB genes were examined using qRT-PCR. Data analysis showed the significant increase in expression levels of all studied genes in the B19V-transfected cells (P < 0.05). Although further researches are required, our findings for the first time suggest the importance of B19V infection to establish an inflammatory microenvironment in the bone marrow and its involvement in inflammation-related diseases. Finally, based on our results, molecular assay to diagnose B19V infection of BM-MSCs prior to stem cell therapy is strongly recommended.

Persistent injury-associated anemia: the role of the bone marrow microenvironment

BackgroundThe regulation of erythropoiesis involves hematopoietic progenitor cells, bone marrow stroma, and the microenvironment. Following severe injury, a hypercatecholamine state develops that is associated with increased mobilization of hematopoietic progenitor cells to peripheral blood and decreased growth of bone marrow erythroid progenitor cells that manifests clinically as a persistent injury-associated anemia. Changes within the bone marrow microenvironment influence the development of erythroid progenitor cells. Therefore, we sought to determine the effects of lung contusion, hemorrhagic shock, and chronic stress on the hematopoietic cytokine response. Materials and methodsBone marrow was obtained from male Sprague–Dawley rats (n = 6/group) killed 7 d after lung contusion followed by hemorrhagic shock (LCHS) or LCHS followed by daily chronic restraint stress (LCHS/CS). End point polymerase chain reaction was performed for interleukin-1β, interleukin-10, stem cell factor, transforming growth factor-β, high-mobility group box-1 (HMGB-1), and B-cell lymphoma-extra large. ResultsSeven days following LCHS and LCHS/CS, bone marrow expression of prohematopoietic cytokines (interleukin-1β, interleukin-10, stem cell factor, and transforming growth factor-β) was significantly decreased, and bone marrow expression of HMGB-1 was significantly increased. B-cell lymphoma-extra large bone marrow expression was not affected by LCHS or LCHS/CS (naïve: 44 ± 12, LCHS: 44 ± 12, LCHS/CS: 37 ± 1, all P > 0.05). ConclusionsThe bone marrow microenvironment was significantly altered following severe trauma in a rodent model. Prohematopoietic cytokines were downregulated, and the proinflammatory cytokine HMGB-1 had increased bone marrow expression. Modulation of the bone marrow microenvironment may represent a therapeutic strategy following severe trauma to alleviate persistent injury-associated anemia.

Governing roles for Trib3 pseudokinase during stress erythropoiesis.

In response to anemia, the heightened production of erythropoietin (EPO) can sharply promote erythroid progenitor cell (EPC) formation. Specific mediators of such EPO- accelerated erythropoiesis, however, are not well understood. Presently, we first report that the expression of Trib3 in adult bone marrow EPCs invivo is nominal at steady state, but strongly activated on EPO challenge. In a knockout mouse model, Trib3 disruption modestly increased steady-state erythrocyte numbers and decreased mean corpuscular volume. Following 5-fluorouracil myeloablation, however, rebound red blood cell production and hemoglobin levels were substantially (and selectively) compromised in Trib3-/- mice versus Trib3+/+ congenic controls. Erythrocytes from 5-fluorouracil-treated Trib3-/- mice additionally were more prone to lysis and exhibited elevated peroxide-induced reactive oxygen species. Exvivo, the development of CD71posTer119pos erythroblasts from Trib3-/- bone marrow progenitors was attenuated, and this was associated with heightened EPO-dependent Erk1/2 activation and moderately increased Akt activation. For developmentally staged EPCs, gene profiling provided further initial insight into candidate mediators of EPO-induced Trib3 gene expression, including Cebp-beta, Atf4, Egr-1, and Nab1. Overall, Trib3 is indicated to act as a novel EPC-intrinsic governor of stress erythropoiesis.

Clonidine reduces norepinephrine and improves bone marrow function in a rodent model of lung contusion, hemorrhagic shock, and chronic stress

Propranolol has been shown previously to restore bone marrow function and improve anemia after lung contusion/hemorrhagic shock. We hypothesized that daily clonidine administration would inhibit central sympathetic outflow and restore bone marrow function in our rodent model of lung contusion/hemorrhagic shock with chronic stress. Male Sprague-Dawley rats underwent 6days of restraint stress after lung contusion/hemorrhagic shock during which the animals received clonidine (75μg/kg) after the restraint stress. On postinjury day 7, we assessed urine norepinephrine, blood hemoglobin, plasma granulocyte colony stimulating factor, and peripheral blood mobilization of hematopoietic progenitor cells, as well as bone marrow cellularity and erythroid progenitor cell growth. The addition of clonidine to lung contusion/hemorrhagic shock with chronic restraint stress significantly decreased urine norepinephrine levels, improved bone marrow cellularity, restored erythroid progenitor colony growth, and improved hemoglobin (14.1±0.6 vs 10.8±0.6g/dL). The addition of clonidine to lung contusion/hemorrhagic shock with chronic restraint stress significantly decreased hematopoietic progenitor cells mobilization and restored granulocyte colony stimulating factor levels. After lung contusion/hemorrhagic shock with chronic restraint stress, daily administration of clonidine restored bone marrow function and improved anemia. Alleviating chronic stress and decreasing norepinephrine is a key therapeutic target to improve bone marrow function after severe injury.

Identification of CAR As a Novel Mediator of Erythroid Differentiation and Migration That Is Specifically Downregulated in Erythropoietic Progenitor Cells in Patients with MDS

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by peripheral cytopenia, an accumulation of dysplastic cells in the bone marrow (BM) and a high risk of transformation to acute myeloid leukemia. Transfusion-dependent anemia develops in most patients suffering from MDS. In patients with refractory anemias the accumulation of dysplastic erythroid BM progenitor cells (EryPC) which paradoxically accompanies peripheral anemia, is a typical finding. Although various hypotheses have been discussed, the biochemical basis of the maturation defect of erythroid cells and their abnormal accumulation in the BM in MDS remains unknown. In order to learn more about abnormally regulated and functionally relevant genes expressed in EryPC, we have recently established a screening program involving mRNA expression profiling studies of EryPC in patients with low-risk MDS (IPSS-R score<7) and control BM samples obtained from patients with other BM neoplasms as well as patients with unexplained cytopenia, normal BM (staging for lymphomas) or reactive/deficiency cytopenias. EryPC were defined as CD45low/CD105+ cells and purified from BM mononuclear cells (MNC) by multicolor flow cytometry (MFC) and cell sorting (purity>95%). mRNA expression profiles were analyzed by Affymetrix array technology (GeneChip U133 Plus 2.0 arrays) and confirmed for a panel of selected mRNA species by qPCR. In mRNA- and MFC-validation experiments, we found that the major Coxsackie-Adenovirus Receptor (CAR) is markedly and specifically down-regulated in CD45low/CD105+ EryPC in patients with MDS when compared to EryPC in normal BM or other control cohorts. In line with this observation, the immature erythroblastic cell lines HEL, K562 and KU812 all stained negative for CAR in flow cytometry analyses. Lentiviral transduction of the full-length CAR gene into these cells resulted in a significantly increased expression of various erythroid differentiation antigens, including CD36, CD71 and CD235a (Glycophorin-A) as determined by flow cytometry and qPCR. As assessed by flow cytometry, the levels of CD235a increased to 158±46% in HEL cells, to 211±23% in K562 cells and to 170±30% in KU812 cells after CAR transduction compared to the empty vector control (p<0.05). In addition, CAR transduction resulted in an increased migration of HEL cells against a serum protein-gradient in a transwell assay (empty vector control: 185±67% vs CAR-transfected cells: 356±74%, p<0.05). Transfection with truncated variants of CAR did not result in an increased expression of erythroid antigens or an increased directed migration. In conclusion, our data show that CAR is a functionally relevant antigen that promotes the expression of early erythroid differentiation antigens on myeloid progenitor cells and their migration against blood serum proteins. In patients with MDS, CAR is specifically downregulated on EryPC, which may have clinical implications both in term of the pathogenesis of the disease and the application of this novel marker in diagnostic MFC algorithms. With regard to functional consequences, we hypothesize that CAR-deficiency is pathogenetically relevant as it may not only contribute to the maturation-defect of erythroid progenitor cells in MDS EryPC but also to the related accumulation of erythroid cells in the BM that is accompanying the peripheral anemia in these patients. DisclosuresNo relevant conflicts of interest to declare.

Erythropoiesis- and Thrombopoiesis-Characterizing Parameters in Adenosine A3 Receptor Knock-Out Mice

Influence of the regulatory system mediated by adenosine A(3) receptors on the functioning of erythropoiesis and thrombopoiesis was studied by means of evaluation of the numbers and attributes of peripheral blood erythrocytes and platelets, as well as of erythroid bone marrow progenitor cells in adenosine A(3) receptor knock-out (Adora3(tm1Jbsn)/Adora3(tm1Jbsn), A(3)AR((-/-))) mice and their wild-type C57BL/6 counterparts, both males and females. Minor but statistically significant disturbances in the properties of erythrocytes, namely in the parameters of mean erythrocyte volume and mean erythrocyte hemoglobin were observed in A(3)AR((-/-)) mice. In addition, adenosine A(3) receptor knock-out mice were found to exhibit an expressive, statistically significant decrease of their blood platelet count, amounting to 17 % and 21 % in males and females, respectively. This decrease in platelet levels was accompanied by a significant 17 % decline in the plateletcrit in both sexes. The obtained data can help to define therapeutic applications based on the principle of adenosine receptor signaling.

Induction of human fetal hemoglobin expression by adenosine-2’,3’-dialdehyde

BackgroundPharmacologic reactivation of fetal hemoglobin expression is a promising strategy for treatment of sickle cell disease and β-thalassemia. The objective of this study was to investigate the effect of the methyl transferase inhibitor adenosine-2’,3’-dialdehyde (Adox) on induction of human fetal hemoglobin (HbF) in K562 cells and human hematopoietic progenitor cells.MethodsExpression levels of human fetal hemoglobin were assessed by northern blot analysis and Real-time PCR. HbF and adult hemoglobin (HbA) content were analyzed using high-performance liquid chromatography (HPLC). DNA methylation levels on human gamma-globin gene promoters were determined using Bisulfite sequence analysis. Enrichment of histone marks on genes was assessed by chromosome immunoprecipitation (ChIP).ResultsAdox induced γ-globin gene expression in both K562 cells and in human bone marrow erythroid progenitor cells through a mechanism potentially involving inhibition of protein arginine methyltransferase 5 (PRMT5).ConclusionsThe ability of methyl transferase inhibitors such as Adox to efficiently reactivate fetal hemoglobin expression suggests that these agents may provide a means of reactivating fetal globin expression as a therapeutic option for treating sickle cell disease and β-thalassemia.

The role of WDR5 in silencing human fetal globin gene expression

Histone H3 lysine 4 (K4) methylation has been linked with transcriptional activity in mammalian cells. The WD40-repeat protein, WDR5, is an essential component of the MLL complex that induces histone H3 K4 methylation, but the role of WDR5 in human globin gene regulation has not yet been established. To study the role of WDR5 in human globin gene regulation, we performed knockdown experiments in both K562 cells and primary human bone marrow erythroid progenitor cells (BMC). The effects of WDR5 knockdown on γ-globin gene expression were determined. Biochemical approaches were also employed to investigate WDR5 interaction molecules. Chromosomal marks in the globin locus were analyzed by ChIP. We found that WDR5 interacted with protein arginine methyltransferase 5 (PRMT5), a known repressor of γ-globin gene expression, and was essential for generating tri-methylated H3K4 (H3K4me3) at the γ-globin promoter in K562 cells. Enforced expression of WDR5 in K562 cells reduced γ-globin gene expression, whereas knockdown of WDR5 increased γ-globin gene expression in both K562 cells and primary human bone marrow erythroid progenitor cells. Consistent with this, both histone H3 and H4 acetylation at the γ-globin promoter were increased, while histone H4R3 and H3K9 methylation were decreased, in WDR5 knockdown cells compared to controls. We found that WDR5 interacted with HDAC1 and a PHD domaincontaining protein, ING2 (inhibitor of growth), an H3K4me3 mark reader, to enhance γ-globin gene transcriptional repression. In human BMC, levels of WDR5 were highly enriched on the γ-promoter relative to levels on other globin promoters and compared to the γ-promoter in cord blood erythroid progenitors, suggesting that WDR5 is important in the developmental globin gene expression program. Our data are consistent with a model in which WDR5 binds the γ-globin promoter in a PRMT5-dependent manner; H3K4me3 induced at the γ-globin promoter by WDR5 may result in the recruitment of the ING2-associated HDAC1 component and consequent silencing of γ-globin gene expression.

Identification of TROP2 (TACSTD2), an EpCAM-Like Molecule, as a Specific Marker for TGF-β1-Dependent Human Epidermal Langerhans Cells

Langerin (CD207) expression is a hallmark of epidermal Langerhans cells (LCs); however, CD207(+) cells comprise several functional subsets. Murine studies showed that epidermal, but not dermal, CD207(+) cells require transforming growth factor-β 1 (TGF-β1) for development, whereas human data are lacking. Using gene profiling, we found that the surface molecule TROP2 (TACSTD2) is strongly and rapidly induced during TGF-β1-dependent LC commitment of human CD34(+) hematopoietic progenitor cells or monocytes. TROP2 is conserved between mouse and human, and shares substantial amino-acid identity with EpCAM, a marker for murine epidermal LCs. To our knowledge, neither TROP2 nor EpCAM expression has been analyzed in human dendritic cell (DC) subsets. We found that (i) all human epidermal LCs are TROP2(+)EpCAM(+); (ii) human dermis lacks CD207(+)EpCAM(-) or CD207(+)TROP2(-) DCs, i.e., equivalents of murine dermal CD207(+) DCs; and (iii) pulmonary CD207(+) cells are TROP2(-)EpCAM(-). Moreover, although EpCAM was broadly expressed by pulmonary and intestinal epithelial cells, as well as by bone marrow erythroid progenitor cells, these cells lacked TROP2. However, although TROP2 is expressed by human LCs as well as by human and murine keratinocytes, most murine LCs, except of a small subset, lacked TROP2. Therefore, TROP2 is a marker for human TGF-β1-dependent epidermal LCs.

The Effects of Environmental Cues on Hematopoietic Stem Cell Colony Fetal Hemoglobin Production.

AbstractAbstract 3860 Objective.Increased fetal hemoglobin (HbF) levels are associated with increased life span and reduced pain crises in patients with Sickle Cell disease. An increased understanding of the mechanisms controlling HbF expression would be important to develop new therapies to increase HbF. The objective of these experiments was to test the hypothesis that interactions between bone marrow (BM) erythroid progenitor cells and the stromal microenvironment influence HbF expression. Materials and Methods.Baboon CD34+ BM cells were harvested and purified by immunomagnetic column chromatography using the 12.8 anti-CD34 mouse monoclonal antibody and immunomagnetic microbeads conjugated to rat anti-mouse IgM (Miltenyi). CD34+ BM Cells were grown in liquid cultures in Iscove's media containing 30% fetal bovine serum, 200ng/ml stem cell factor, 2u/ml erythropoietin, and 1uM dexamethasone in the presence of an AFT024 mouse fetal liver cell line and an OP9 mouse bone marrow cell line as feeder layers, in methylcellulose media, and in liquid media. Globin chain synthesis in cultures was measured on d11 and d14 by biosynthetic radiolabeling. Cells were incubated overnight in leucine-free α-MEM media containing 50uCi/ml [3H leucine]. Globin chains were separated by High Performance Liquid Chromatography (HPLC), and the radioactivity in each fraction was determined by liquid scintillation counting. To correlate changes in γ-globin expression with DNA methylation of the γ-globin gene promoter, the methylation state of 5 CpG sites within the γ-globin promoter region was determined by bisulfite sequencing. Erythroid cells were purified from cultures on d14 by immunomagnetic column chromatography using mouse anti-baboon RBC monoclonal antibody. DNA was isolated from purified erythroid cells using Qiagen kits. Bisulfite modification was performed using Epitect bisulfite kits. Two rounds of PCR amplification were performed using nested primers flanking 5 CpG residues within the baboon γ-globin gene promoter. Minilysate DNA was prepared from at least 10 independent clones for each sample. Sequence analysis of purified DNA samples was performed at the University of Illinois DNA Sequence facility. Results.Elevated levels of γ-globin chain synthesis were observed in cells cultured in methylcellulose and liquid media in the absence of a feeder layer when compared to cells grown in the presence of feeder layers. On d11, the γ/γ+β chain synthetic ratio was 0.623 in cells cultured in methylcellulose and 0.324 in cells cultured in liquid media, compared to 0.092 in cells cultured in the presence of AFT024 feeder layers and 0.178 in cells cultured in the presence of OP9 feeder layers. On d14, the γ/γ+β chain synthetic ratio was 0.663 in cells cultured in methylcellulose and 0.349 in cells cultured in liquid media, compared to 0.135 in cells cultured in the presence of AFT024 feeder layers and 0.252 in cells cultured in the presence of OP9 feeder layers. The level of DNA methylation (%dmC) of 5 sites in the γ-globin gene promoter negatively correlated with levels of HbF production. On d14, the level of DNA methylation was 79% in cells cultured in methylcellulose, 72% in cells cultured in liquid media, and 97% in cells grown in the presence of AFT024 feeder layers. Conclusions.Cells grown in the presence of AFT024 feeder layers expressed physiologic levels of γ-globin and cells grown in the presence of OP9 feeder layers expressed midrange levels of γ-globin. Significantly increased γ-globin expression was observed in cells cultured in the absence of a feeder layer. The γ-globin promoter in cells grown in the presence of the AFT024 feeder layer exhibited a significantly higher degree of methylation than in cells grown in methylcellulose and liquid media. These results show that interactions between erythroid progenitor cells and the stromal microenvironment can influence both the level of γ-globin expression and the DNA methylation of the γ-globin gene promoter. These results have clinical relevance; if the bone marrow microenvironment could be effectively altered in vivo, methylation of the γ-globin promoter could be decreased and HbF production increased. Further experiments must be performed to determine what is mediating the methylation of the γ-globin promoter when cells are grown on these feeder layers. Disclosures:No relevant conflicts of interest to declare.

Decline of the Red Blood Cell Count in Patients Receiving Androgen Deprivation Therapy for Localized Prostate Cancer: Impact of ADT on Insulin-like Growth Factor-1 and Erythropoiesis

To elucidate the mechanism of blood hemoglobin loss in patients with prostate cancer during androgen deprivation therapy (ADT), and to examine the activity of the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis during ADT, which plays an important role in hematopoiesis. A total of 83 patients with localized prostate cancer, who received ADT, were prospectively studied on the basis of their blood samples at the baseline and after ADT for 6 months. Before ADT, the IGF-1 level was correlated with the red blood cell (RBC) count (Spearman's rank correlation coefficient analysis [rs]=0.315, P=.011), hemoglobin (rs=0.278, P=.018), and mean corpuscular volume (rs=0.266, P=.020), but such relationships disappeared after ADT. After ADT, the serum IGF-1 level increased compared with that at the baseline (21+/-6 vs 18+/-5 nmol/L, respectively, P<.001), but no change was observed in the serum GH level (P=.691). There was no difference between erythropoietin and interleukin-6 concentrations before and after ADT (P=.852 and P=.208, respectively). The hemoglobin concentration and RBC count declined after ADT compared with those before treatment (P<.001 for each). Although the mean corpuscular volume declined after ADT (P=.002), the mean cell hemoglobin was comparable between before and after ADT (P=.676). Despite the unaffected GH, erythropoietin, and interleukin-6 levels, the serum IGF-1 concentration was elevated by ADT. Even with the increased IGF-1 level, the RBC count and hemoglobin concentration declined after ADT. IGF-1 in the bone marrow erythroid progenitor cells might be functionally inactivated during ADT.

Meloxicam Elevates Serum Concentration of Erythropoietin and Numbers of Bone Marrow Erythroid Progenitor Cells in Sublethally Gamma-Irradiated Mice

Meloxicam, a non-steroidal anti-inflammatory drug selectively inhibiting cyclooxygenase-2, has been found to enhance the regeneration of erythroid progenitor cells (BFU-E) in the femoral bone marrow of mice when administered after sublethal irradiation (4 Gy gamma-rays). In mice treated with meloxicam once daily on days 3, 4, 5, and 6 after irradiation, the values of BFU-E per femur in meloxicam-treated mice were on days 7 and 14 after irradiation at the levels of 156 % and 191 %, respectively, related to those in irradiated saline-treated controls (P &lt; 0.01 and P &lt; 0.001, respectively). Moreover, it has been shown that these effects of meloxicam can be associated with its ability to stimulate erythropoietin production in irradiated mice. Six and 12 hours after one dose of meloxicam given on day 3 after irradiation, the serum level of erythropoietin was twofold higher in comparison with irradiated saline-treated controls (P &lt; 0.05). These findings may have practical implications in the treatment of myelosuppression.

Erythropoietin and outcome prediction in patients with heart failure: the plot thickens...

Heart failure syndromes represent a principal cause of morbidity and mortality in the Western world.1 The importance of measures for diagnosis, surveillance, and treatment of patients with chronic heart failure (CHF) are likely to attract increasing attention as the prevalence of the syndrome will rise with the ageing of the population. One of the most studied clinical determinants of outcome in patients with CHF is haemoglobin level.2 There are many reports showing that anaemia is associated with a reduced quality of life and increased mortality in these patients.2 However, many questions remain that pertain to the precise mechanisms of anaemia, the need to treat it, and the desired haemoglobin goals. Erythropoietin (Epo) is a 34 kDa glycoprotein synthesized by renal peritubular cells, but also by neuronal and hepatic macrophages and other cells.3 The output signals that control Epo homeostasis are principally provided by a set of transcription factors that ‘sense’ changes in oxygen levels and are termed accordingly as hypoxia-inducible factors. The Epo receptor is found most abundantly in bone marrow erythroid progenitor cells, although other tissues also express it. Aside from the well described action of Epo as a proliferative enhancer of erythropoiesis, in recent … *Corresponding author. Tel: +972 3 6974762, Fax: +972 3 546 9832, Email: jacobg{at}post.tau.ac.il

Parvovirus B19 Infection in the Immunocompromised Host

Human parvovirus B19 is a single-stranded DNA virus with a predilection for infecting rapidly dividing cell lines, such as bone marrow erythroid progenitor cells. People with defective cell-mediated immunity (eg, severe combined immunodeficiency syndrome; acquired immunodeficiency syndrome; and patients receiving immunosuppressive therapy, ie, post organ transplant) can develop pure red cell aplasia, in which suppression of erythroid precursors is permanent. Identification of parvovirus inclusions in marrow biopsies and subsequent confirmation of infection by in situ hybridization is important in the assessment of anemia in immunodeficient patients. Our objective is to provide a general overview of the parvovirus B19 infection and its characteristics in immunocompromised patients and to summarize updated information regarding the clinicopathologic features, pathobiology, and laboratory diagnosis of this subject. The pathologist should be aware of the wide spectrum of manifestations of parvovirus B19 infection depending on the patient's hematologic and immunologic status.

PBI-1402: A New Candidate for the Treatment of Anemia.

PBI-1402 is an orally active low molecular weight synthetic compound which is currently in a phase Ib/II clinical trial in patients with anemia associated with cancer and/or chemotherapy. PBI-1402 stimulates the in vitro/ex vivo proliferation and maturation of hematopoietic progenitors (erythroid and myeloid populations) with an activity comparable to EPO. Furthermore, an additive effect is observed in combination with EPO. PBI-1402 enhances the differentiation of pluripotent stem cells: CFU-GEMM, CFU-GM with a predominant effect on BFU-E. Clinical phase I study showed that PBI-1402 is devoid of significant side effects. In addition, the clinical phase I results showed statistical increase (100%, p<0.0001) of relative and absolute reticulocyte count in healthy volunteers after 21 days of oral treatment compared to placebo. Preclinical results demonstrated that PBI-1402 yields an increase in erythrocytes in the systemic circulation. In myeloblated mice that received a syngeneic bone marrow transplant, oral treatment with PBI-1402 resulted in a significant increase in peripheral erythrocyte count, hemoglobin, platelet and bone marrow erythroid progenitor cells. PBI-1402 is targeted as an adjunct to cytotoxic drugs, radiotherapy and bone marrow transplantation for the treatment of anemia.

Transgenic GATA1ie3.9int - DAPK2 Mice Exhibit Low-Grade Anemia, Deficient Stress Erythropoiesis, and Compromised Erythroblast Survival.

Death-associated protein kinases (DAPKs) include DAPK-1, DAPK-2, ZIPK and death receptor associated kinase 1 & 2 (DRAK-1 and -2). DAPK-1 and -2 are highly homologous in their shared serine-threonine kinase domains, and calmodulin and phospho-serine regulatory regions. DAPK1 is cytoskeletally associated via ankyrin repeats, and modulates a p19ARF/p53 pathway. DAPK2 is cytoplasmic, less widely distributed, and is expressed predominantly in erythroid cells. Hypothesized effects of DAPK2 on erythroblast formation presently were tested by preparing and studying several independent lines of GATA1ie3.9int-DAPK2 transgenic mice. At steady-state, hematocrits in GATA1ie3.9int-DAPK2 mice were mildly yet significantly decreased (while other lineages were essentially unaffected). Reticulocyte production also was modestly increased, and spleen morphologies were altered. During hemolytic anemia, however, DAPK2 mice exhibited comparably sharp deficiencies in stress erythropoiesis including prolonged decreases in hematocrits (as observed in repeated experiments at varied phenylhydrazine dosing). The basis for this stress-associated erythropoietic defect due to DAPK2 was investigated further in primary bone marrow erythroid progenitor cells ex vivo. A stage-specific defect in erythroblast formation was discovered specifically at KitnegCD71highTer119neg stage. Later stage CD71highTer119pos DAPK2-erythroblasts also were somewhat under-represented (but perhaps as a consequence of decreased KitnegCD71high pools.) In addition, DAPK2-progenitors gave rise to increased frequencies of Annexin V positive apoptotic erythroblasts. These first-time analyses of DAPK2 function in a relevant primary cell model reveal negative regulation of stage-specific erythroblast pool selectively during stress erythropoiesis, and in part via pro-apoptotic mechanisms.

Prevalence of the Activating JAK2 Tyrosine Kinase Mutation V617F in the Budd-Chiari Syndrome.

Budd-Chiari Syndrome (BCS) is a group of disorders resulting from obstruction to hepatic venous outflow; myeloproliferative disorder (MPD) accounts for 10–40% of cases. A number of BCS cases labelled as ‘idiopathic' do not fulfil the accepted diagnostic criteria for MPD but have features suggestive of a latent form of MPD based on hyperplastic bone marrow and spontaneous erythroid colony progenitor cell culture studies; these cases may subsequently develop overt MPD. A clonal mutation in JAK2 tyrosine kinase (JAK2V617F) occurs in a high proportion of patients with MPD and is of potential use in the characterization of latent MPD in BCS. We studied 44 patients with BCS (female n=27, mean age 36.1 years, SD 13) presenting between 1985 and 2005. Genomic DNA obtained from archived bone marrow films was screened by allele-specific PCR for the JAK2V617F tyrosine kinase mutation. JAK2V617F was detected in 27/44 (61.4%) subjects. Fulminant hepatic failure was the presenting feature in 86.4%. 3/38 subjects had previously diagnosed Polycythemia Vera and all of these were JAK2V617F positive. Analysis for hereditary thrombophilia in 32/44 cases showed Factor V Leiden (FVL) heterozygosity (n=2) and Protein C deficiency (n=1); JAK2V617F was detected in one of the FVL cases. Screening for antiphospholipid antibodies and paroxysmal nocturnal hemoglobinuria in 31 and 30 out of 44 cases respectively proved negative. 22/44 cases had splenomegaly of which 72.2% had JAK2V617F detected. Mean hemoglobin concentration was higher in patients with JAK2V617F (12.4 g/dL, SD 2.8) compared to the wild-type allele (10.2 g/dL, SD 2.1)(p=0.005). Mean neutrophil count was similar (p=0.26) in both groups (JAK2V617F 7.8 x109/L, SD 5.5; wild-type 6.1 x109/L, SD 4.5). Mean platelet count was higher (p=0.01) in subjects with JAK2V617F (289 x109/L, SD 192) compared to wild-type (180 x109/L, SD 158). The bone marrow (BM) was hyperplastic in all lineages in 19/44 subjects of which 15 (78.9%) contained the JAK2V617F mutation. Importantly, in 25/44 subjects without hyperplastic BM, 48% had JAK2V617F detected. Clonal cytogenetic abnormalities occurred in 6/44 subjects all of which carried JAK2V617F. In 35 evaluable subjects, 10/35 (28.6%) showed endogenous erythroid colony (EEC) formation (8/10 JAK2V617F positive), and the remainder (25/35) showed no EEC formation (14/25 JAK2V617F positive). Treatment of BCS included anticoagulation (n=44), porto-systemic shunt insertion (n=26) and orthotopic liver transplantation (OLT) (n=15). Overall survival was 81.8% (median 32.5 months, range 2 days-240 months). In the 8 non-survivors, JAK2V617F was detected in 50%. 15/44 subjects underwent OLT (11/15 JAK2V617F positive) of which 14/15 are alive at a median of 36 months post transplant (1–240 months). 14/44 subjects were treated with cytoreductive treatment following diagnosis of BCS and in 92.9% of these JAK2V617F was detected. 5/15 subjects went on to receive cytoreductive treatment following OLT of which all were JAK2V617F positive. We have found a high prevalence of JAK2V617F in patients with BCS. Latent MPD was missed in 12/25 subjects on BM morphology and 14/25 subjects on BM progenitor culture. We suggest that JAK2V617F analysis should be performed in all new cases of BCS. Given their age, these patients are possible candidates for JAK2 targeted therapy.

Stromal Cell-Derived Factor-1/CXCL12 Selectively Counteracts Inhibitory Effects of Myelosuppressive Chemokines on Hematopoietic Progenitor Cell Proliferation In Vitro

A variety of cytokines and chemokines exert potent myelosuppressive effects that play a role in the maintenance of hematopoiesis, which, if unchecked, may result in pathological impairment of blood cell production. Processes that modulate these myelosuppressive effects are not well defined. Here we demonstrate that stromal cell-derived factor-1 (SDF-1/CXCL12), known for its ability to attract and to promote survival of hematopoietic progenitor cells (HPCs) and stem cells, blocks the effects of a broad range of myelosuppressive chemokines on proliferation of HPCs in vitro. The regulatory effects of SDF/CXCL12 on colony formation by mouse bone marrow granulocyte-macrophage (CFUGM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells were assessed. These cells were stimulated to proliferate by combinations of growth factors, such that responses of immature HPCs could be assessed. SDF-1/CXCL12 potently blocked myelosuppressive responses induced by CCL2/MCP-1, CCL3/MIP-1alpha, CCL19/CKbeta-11, CCL25/TECK, CXCL4/PF4, CXCL8/IL-8, CXCL10/IP-10, and XCL1/Lymphotactin. However, SDF/CDL12 did not influence myelosuppression induced by tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta or the iron-binding proteins H-ferritin or lactoferrin (LF). LF, previously shown to suppress release of growth factors, is shown here to also suppress proliferation of immature subsets of HPCs. HPCs from marrows of mice expressing an SDF-1/CXCL12 transgene were insensitive to inhibition by SDF/CXCL12-sensitive myelosuppressive chemokines, but not to SDF/CCL12-insensitive cytokines (TNF-alpha, IFN-gamma, TGF-beta, H-Ferritin, or LF). Thus, SDF-1/CXCL12 differentially and selectively regulates suppression of HPC proliferation by chemokines. These effects may counter myelosuppressive effects of certain chemokines in vivo, where proliferation of HPCs must be sustained.