Peripheral Red Cells Research Articles

Evaluation of (–)-α-Bisabolol Toxicity in Drosophila melanogaster and Human Blood Peripheral Mononuclear and Red Cells

Evaluation of (–)-α-Bisabolol Toxicity in Drosophila melanogaster and Human Blood Peripheral Mononuclear and Red Cells

Nonerythropoietic Erythropoietin Mimetic Peptide ARA290 Ameliorates Chronic Stress-Induced Depression-Like Behavior and Inflammation in Mice

Major depressive disorder (MDD) is a highly prevalent psychiatric disorder. But the treatment of depression remains challenging. Anti-inflammatory treatments frequently produce antidepressant effects. EPO-derived helix-B peptide ARA290 has been reported to retain the anti-inflammatory and tissue-protective functions of EPO without erythropoiesis-stimulating effects. The effects of ARA290 on MDD remain elusive. This study established chronic unpredictable mild stress and chronic social defeat stress mouse models. Daily administration of ARA290 during chronic stress induction in two mouse models ameliorated depression-like behavior, similar to fluoxetine. With marginal effects on peripheral blood hemoglobin and red cells, ARA290 and fluoxetine reversed chronic stress-induced increased frequencies and/or numbers of CD11b+Ly6Ghi neutrophils and CD11b+Ly6Chi monocytes in the bone marrow and meninges. Furthermore, both drugs reversed chronic stress-induced microglia activation. Thus, ARA290 ameliorated chronic stress-induced depression-like behavior in mice through, at least partially, its anti-inflammatory effects.

Methotrexate Polyglutamate Values in Children and Adolescents With Acute Lymphoblastic Leukemia During Maintenance Therapy.

Inadequate adherence to maintenance therapy is a major cause of relapse in patients with acute lymphoblastic leukemia (ALL). Therapeutic monitoring of mercaptopurine (thiopurine) red cell metabolites to assess adherence has been available for many years. Recently a clinical laboratory improvement amendments of 1988-approved test for methotrexate with three polyglutamate residues (MTXPG3) measured in peripheral blood red cells was approved. MTXPG3 is the primary intracellular metabolite of methotrexate, and like thiopurine metabolites, is retained for the life of the red cell giving an estimate of drug exposure over time. Normative values for MTXPG3 are available for adults and children with rheumatoid arthritis on methotrexate monotherapy, which are not applicable for patients with ALL on maintenance. Older literature on the MTXPG3 fraction in children with ALL is limited. We examined the MTXPG3 levels from 123 samples in 76 patients with ALL on maintenance oral methotrexate and mercaptopurine that were collected for clinical care. Male individuals had significantly higher MTXPG3 levels than female individuals which was unrelated to absolute neutrophil count, age, serum creatinine, and average doses of methotrexate or mercaptopurine. The MTXPG3 5th, 10th, 90th, and 95th percentile values are 0, 8.4, 53, and 64, respectively with a median of 24.7 nmol/L. The low 5th percentile MTXPG3 reflects 6 samples from 3 patients, age 16 to 21 years that were considered poorly adherent before collecting the specimen. As with red cell thiopurine (mercaptopurine) metabolites, MTXPG3 normative values may provide useful information to monitor for poor patient adherence or methotrexate toxicity during maintenance chemotherapy in ALL.

Analysis of ABO chimera from peripheral red cells and reticulocytes by flow cytometry and micro gel column technique in patients post-ABO-incompatible HSCT.

How to choose an appropriate method to monitoring dynamic ABO chimera post-ABO-incompatible HSCT is crucial to not only assess the status of erythroid engraftment but also achieve personalized safety transfusion. We evaluated the efficacy of micro gel column technique (MGCT) and flow cytometry (FCM) by series of artificial ABO chimera mixtures from 0.5% to 50% and by investigating 15 cases of ABO-incompatible HSCT patients with longitudinally ABO blood grouping and ABO chimera from RBCs and reticulocytes. 5% and 2% of ABO chimera mixtures can be efficiently detected by MGCT and FCM, respectively. 6.3% of donor RBCs with 44.77% of donor reticulocytes in the early phase and 7.9% of patient RBCs with 96% of donor reticulocytes in the phase of complete donor type can be detected by FCM rather than failure by MGCT simultaneously. However, in case 8#, 8.6% of donor reticulocytes rather than 99.1% of donor RBCs on the 98th day post-HSCT could adequately predict early relapse. Investigation of ABO chimera from reticulocytes by FCM is a more effective strategy rather than ABO blood grouping and RBCs by FCM to indicate the true progress of erythroid alteration and achieve personalized safety transfusion post-ABO-incompatible HSCT.

Loss of NRF2 Function Worsens the Pathophysiology of Sickle Cell Disease in a Transgenic Mouse Model

The basic leucine zipper transcription factor, nuclear factor (erythroid-derived 2)-like 2 (NRF2) plays a critical role in the cellular antioxidant response to control oxidative stress. We and others previously demonstrated that NRF2 activation enhances γ-globin gene transcription and fetal hemoglobin expression in human primary erythroid progenitors through changes in chromatin structure (Zhu et al., Haematologica 2017). In this study, we investigated the protective role of NRF2 in reversing the pathophysiology of sickle cell disease (SCD) in a SCD/NRF2 knockout (SCD/NRF2KO) transgenic model created in our lab by crossbreeding Townes SCD mice (Ryan et al., Science 1997) and NRF2 knockout mice (Kuroha et al., J Biochem 1998). The NRF2 gene is transmitted through autosomal recessive Mendelian genetics for wild-type, heterozygote and NRF2KO pups. By contrast, the SCD/NRF2KO genotype was present in <2% of pups. In addition, the fertility and litter size of SCD/NRF2KO females were lower than SCDWT mice, suggesting a critical role of NRF2 in the survival of pups during gestation. To determine the hematopoietic effect of NRF2KO in SCD, we monitored the complete blood count with differential and reticulocyte count. There was no significant change in any parameters except higher total white blood cell counts in the SS/NRF2KO mouse suggesting increased inflammation. Examination of globin gene expression patterns in SS/NRF2KO mice showed reduced γ-globin gene expression during erythroid differentiation in the E13.5 and E18.5 fetal liver, adult spleen and bone marrow. In addition, peripheral blood red cells had a 33% increase (p<0.05) in reactive oxygen species and a significant 38% increase in sickling under in vitro hypoxic conditions. We next characterized the effects of NRF2 loss on organ pathology. The SCD/NRF2KO mice displayed greater splenomegaly indicating exaggerated hemolysis most likely due to high levels of reactive oxygen species. By 8-10 weeks of age, the SCD/NRF2KO mouse showed significant inflammation by hematoxylin-eosin staining of the spleen, lung and liver when compared to SCD/NRF2WT mice. Protein expression profiling by western blotting using adult spleen demonstrated downregulation of the antioxidant proteins heme oxygenase 1 (HMOX1), NADPH: quinone oxidoreductase 1 (NQO1) and catalase by 31%, 60%, and 48% respectively (p<0.05). To further support a severe disease phenotype, the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were increased by 1.7-fold and 2.3-fold (p<0.05) while vascular endothelial growth factor (VEGF) levels were not changed. Lastly, the expression of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), monocyte chemoattractant protein (MCP-1) and macrophage migration inhibitory factor (MIF-1) was elevated in SCD/NRF2KO mice compared to SCDWT mice. These data validate a critical role of NRF2 in ameliorating the phenotypic severity of SCD by protecting against red blood cell sickling, oxidative stress and tissue inflammation. Furthermore, the ability of NRF2 to mediate fetal hemoglobin induction provides a rationale for the development of therapeutic agents that activate NRF2 expression. Disclosures No relevant conflicts of interest to declare.

Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine β-thalassemia

AbstractIn β-thalassemia, unequal production of α- and β-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of β-thalassemia intermedia (Hbbth1/th1 mice) to investigate effects of a modified activin receptor type IIB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbbth1/th1 mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbbth1/th1 mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-β superfamily in late-stage erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE.

Treatment With Minihepcidin Peptide Improves Anemia and Iron Overload In a Mouse Model Of Thalassemia Intermedia

IntroductionBeta-thalassemia is a inherited disorder characterized by mutations in the beta-globin gene that lead to reduced or absent beta-globin synthesis. As a consequence, in erythroid cells there is a relative excess of alpha globin chains and heme forming toxic hemichromes, which lead to oxidative damage, impaired erythroid cell maturation, premature cell death and anemia. Under these conditions, the anemia and the elevated erythropoietin levels increase the proliferation of the erythroid progenitors, suppressing the expression of the iron master regulator hepcidin. Hepcidin targets the iron exporter ferroportin present on enterocytes and macrophages. High levels of hepcidin limit iron absorption and iron recycling but low levels of hepcidin increase iron absorption. Chronic low levels of hepcidin, as in β-thalassemia, eventually lead to iron overload. The th3/+ mouse model of thalassemia intermedia (Non-Transfusion Dependent Thalassemia or NTDT), reproduces the main features of the disease including anemia, increased erythropoiesis, splenomegaly and iron overload due to low levels of hepcidin. It has been previously demonstrated that modestly increasing hepcidin levels in these mice ameliorates both anemia and iron overload (Gardenghi et al, JCI, 2010). MethodsWe used minihepcidin M004, a modified peptide analog of the 9 N-terminal amino acids of hepcidin, to reproduce the biological effects of hepcidin. In a dose-ranging study, we treated th3/+ animals with daily intraperitoneal (ip) injections of M004 at doses of 6.25-100 µg/day for 2-6 weeks or with subcutaneous (sc) dosing of 52.5 μg twice a week for 6 weeks. ResultsIn animals treated with the lower ip doses of M004 (6.25-25 μg/day for 2-4 weeks), mild iron restriction was observed (Tf sat ≥15%). Flow cytometry studies using the markers CD44 and Ter119 demonstrated an increase in the proportion of mature erythroid cells. Consistent with the flow cytometry findings, a 30% increase in peripheral red cells (rbc) was observed associated with a reduction in reticulocyte count (>62%) and a reduction in spleen size (>40%), reflecting improved erythropoietic efficiency. At the 12.5 and 25 µg doses, Hb level increased by 1.8-2.5 g/dL. Longer treatment (6 weeks) with 12.5-25 µg/day led to greater iron restriction and the loss of therapeutic effect at 6 weeks. In the same animals, measurement of total organ iron concentrations demonstrated no changes in the liver and kidney, but a 4-fold iron increase in the spleen. Prussian blue staining confirmed increased sequestration of iron in splenic macrophages and Kupffer cells.At high ip doses of M004 (100 μg/day for 2 weeks) the mice developed severe iron restriction (Tf sat = 6%) and exacerbation of anemia (Hb reduction of 4.5 g/dL). Accumulation of immature erythroid precursors was observed by flow cytometry studies, reflecting the erythroid maturation block caused by lack of iron for hemoglobin generation.Subsequent pharmacokinetic studies in rats indicated that plasma levels of M004 increased gradually with sc dosing and were still increasing 24h after dosing, indicating accumulation of drug levels and iron restriction with daily administration. We therefore explored whether less frequent dosing may be preferable to achieve sustained improvement in anemia and normalization of tissue iron. Sc dosing of M004 at 52.5 µg twice weekly was associated with an increase in rbc at 6 weeks to levels observed in WT animals, and an Hb increase of almost 3 g/dL. Reduced spleen weight (-51%) and reticulocyte count (-61%) reflected improved erythropoietic efficiency, further corroborated by flow cytometry studies. Reduction in hemichrome and ROS formation coupled with improvement in rbc morphology and reduced RDW indicated that hematological improvement was likely caused by improved erythroid cell survival as a result of reduced oxidative damage secondary to decreased erythroid iron uptake. Evaluation of total organ iron concentrations showed a reduction of 77% and 54% in the liver and kidney, respectively. Remarkably, the liver iron values were normalized compared to WT animals. Splenic iron increased only ∼1.5 times or 148%. However, when the total weight of the spleen was taken in account, the total splenic iron was reduced (-39%). ConclusionMinihepcidin peptides may be beneficial for the treatment of beta thalassemia intermedia by improving anemia and reducing tissue iron burden. Disclosures:Casu:Merganser: Research Funding. Goldberg:Merganser: Research Funding. Nemeth:Merganser: Equity Ownership. Ganz:Merganser: Equity Ownership. MacDonald:Merganser : Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rivella:Alexion: Consultancy; Biomarin: Consultancy; Merganser: Equity Ownership, Research Funding; Isis: Consultancy, Research Funding; Bayer: Consultancy; Novartis: Consultancy; Imago: Consultancy.

Functional Hyposplenism in Long-Standing Renal Transplant Recipients

BackgroundA nephrologist with expertise in ultrasonography noticed that patients with longstanding renal grafts had smaller spleens than subjects undergoing initial post-transplantation imaging. This putative finding prompted us to pursue a further investigation into splenic function based on Doppler ultrasound and hematologic parameters. MethodsWe enrolled 47 patients with functioning long-standing kidney grafts, measuring longitudinal diameter of the spleen, hilar and intrasplenic peak systolic velocities (PSV), and hilar and intrasplenic resistivity indices of the splenic artery as well as mean arterial blood pressure (MAP). Giemsa-stained peripheral blood smears were examined for the presence of Howell-Jolly bodies (HJBs) using light microscopy. The patients were then divided into HJB present (HJ+) or absent (HJ−) groups for further comparison. ResultsThe overall mean age of 21 females and 26 males was 47.8 ± 12.0 years, and the mean time after transplantation was 2750 ± 1818 days (range, 208–6446). HJBs were detected in 23/47 patients (48.9%). The intrasplenic artery PSV was significantly lower and MAP higher in the HJ+ group (P < .05). There was no difference in spleen size between the groups. DiscussionHJBs in peripheral blood red cells, an indicator of hyposplenism, was associated with reduced intrasplenic artery PSV, suggesting dysfunction, which may play a role in the known vulnerability of renal transplant recipients to infections.

Metabolic Pathways Control Normal and Beta-Thalassemic Erythroid Cell Maturation

Abstract 369Erythroid cell maturation requires the integration of erythropoietin receptor (EpoR)-mediated signaling pathways and transcriptional programs of erythroid cell proliferation and differentiation largely orchestrated by GATA-1 and its transcriptional partners. Although red blood cells (RBC) purely rely on glycolysis for energy production, and metabolic processes specifically autophagy have been implicated in erythroid maturation, the potential involvement of metabolic pathways in the control of erythroid cell production is not known. Foxo3 transcription factor is a direct target of GATA-1, functionally regulated by EpoR signaling and essential for the redox regulation of erythropoiesis. Mammalian target of rapamycin (mTOR) kinase is a critical regulator of metabolic processes. We have found that generation and cycling of early erythroid precursors is controlled by a redox-mediated Foxo3-mTOR signaling. Terminal erythroid maturation is specifically compromised in Foxo3-deficient mice. Terminal maturation involves nuclear condensation, enucleation, mitochondrial clearance and complete adoption (or conversion) of glycolytic pathway by erythroid cells. We found RBC to be significantly decreased in Foxo3 mutant bone marrow and peripheral blood. This was in contrast to the increase of polychromatophilic erythroblasts associated with an increase in the total TER119+ population in Foxo3 mutant bone marrow likely reflecting a compensatory mechanism. Notably, using DRAQ5, a DNA-binding fluorescent dye, we found the rate of erythroblasts’ enucleation in Foxo3 mutant mice to be significantly compromised. During erythroid maturation Riok3 and Mxi1 transcripts encoding for two important regulators of fetal liver erythroid enucleation are highly upregulated in the bone marrow and robustly expressed in the adult normoblasts and reticulocytes. In agreement with defective enucleation expression of both Riok3 and Mxi1 is highly reduced in Foxo3 mutant erythroblasts and reticulocytes. Interestingly, a two-week in vivo treatment of wild type (WT) and Foxo3−/− mice with rapamycin, a specific inhibitor of mTOR complex 1 (mTORC1) activity, increased significantly the rate of enucleation in a Foxo3-dependent manner, suggesting that mTOR requires Foxo3 activity in supporting erythroid cell maturation. Importantly, targeting mTOR ameliorates beta-thalassemia as inhibition of mTOR signaling by rapamycin treatment improved erythroid cell maturation in the bone marrow, resulted in significant increase in total peripheral blood red cells and hemoglobin (1 to 1.5 g/dl increase) as well as significant reduction in reticulocyte production of beta-thalassemic intermedia (th3/+) mice. Combination of thiazole orange (an RNA and DNA probe) with DRAQ5 determined that in addition to enucleation, the relative production of reticulocytes is also decreased significantly in Foxo3 mutant bone marrow. Strikingly, 6,1% of RBC (CD71 negative) in the peripheral blood of Foxo3−/− animals contained mitochondria (CD71−Mito+) as compared to 0.7 % of wild type RBC. Autophagy is strongly implicated in late stage erythroid cell maturation and mitochondrial removal from reticulocytes. In agreement with a function for Foxo3 in control of mitochondrial removal, expression of Ulk1 (Atg1) and Nix (Bnip3l) both regulators of mitochondrial clearance via autophagy was highly downregulated in Foxo3 mutant normoblasts and reticulocytes. The expression of Nix was notable since Nix was upregulated over 40 fold in wild type but not in Foxo3 mutant reticulocytes as evaluated by the Fluidigm™ microfluidics array technology. These results are consistent with the expression pattern of Foxo3 that is highly upregulated with erythroid maturation and is the most highly expressed in normoblasts. Collectively our results indicate that Foxo3 has a key function in the regulation of terminal erythroid cell maturation. They also suggest that rapamycin may be considered for the treatment of beta-thalassemia. These results are consistent with the model of FOXO3a induction during late human erythroid cell maturation. Based on these studies we propose that Foxo3 coordinates metabolic pathways with the transcriptional program of terminal erythroid cell maturation. Understanding this metabolic program is likely to impact efficient RBC production in culture. Disclosures:Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

Hyposplenism in Long-Standing Renal Transplant Recipients

Background: In our unit, the routine screening for kidney recipients include surveillance with sonography/Doppler after surgery, irrespective of graft function, and at least once a year later on. Almost all studies are performed by an experienced trained nephrologist, who noticed that patients soon after post-transplant showed a larger spleen than patients with long-standing functioning renal graft. These findings, together with the report that in chronic graft versus host disease in bone marrow transplant recipients develop spleen size reduction associated with hyposplenism, and hyposplenism is known to increase susceptibility to infections with encapsulated bacteria, prompted the authors to decide to study splenic function in renal transplant recipients based on sonographic/Doppler and haematological parameters. Methods: The study design was a cross-sectional survey based on single samples. We enrolled two groups: one in the immediate postoperative days (early group) and the other with functioning long-standing kidney graft (late group). To evaluate splenic function, two studies were carried out. First, longitudinal diameter of the spleen and resistivity index of splenic artery at hilum and intrasplenic were measured. All sonograms were performed by a single observer. Second, stained with Giemsa peripheral blood smears (PBS) were reviewed for the presence of Howell-Jolly bodies, using a light microscope viewing. All blood samples were examined by a single observer. Examination of the PBS for Howell-Jolly bodies was restricted to the late group. According to the result, late group was further divided in Howell-Jolly bodies positive (HJ+) or negative (HJ−). Spleen size determination by ultrasonography and Doppler studies were performed in both groups. Immunosuppressive drug regimen used corticosteroid, azathioprine or mycophenolate mofetil and calcineurin inhibitor or mTOR inhibitor. Results: We studied 52 unselected adult patients who had undergone kidney transplantation from a deceased donor and 24 from living donor. The mean age was 48.4±11.5 years (range: 23-74 years). Mean follow up was 11.4±14.2 days for early group (range: 1-68 days) and 2698±1177 days for late group (range: 215-6446 days). Late group had smaller spleen than early group (95.8±16.5 vs 106.3±16.3 mm, p=0.010). Howell-Jolly bodies were found in 52% (24/46) of patients. Intrasplenic resistivity index tended to be higher in HJ+ patients (0.57±0.09 vs 0.51±0.09, p=0.055). There was no correlation between Howell-Jolly detection and immunosuppressive drug regimen. Serum creatinine level was the same despite Howell-Jolly detection or not. Discussion: The presence of Howell-Jolly bodies in the peripheral blood red cells is considered an indicator of hyposplenism. Our findings that a group of transplant patients may develop significant spleen size reduction and detection of Howell-Jolly bodies associated with increased intrasplenic resistivity index suggest splenic dysfunction in these patients, yet the clinical significance of these findings to the outcome of the graft is unknown. Nevertheless, poor splenic function may play a role in the vulnerability to infections in transplant recipients.

Regulation of Erythroid Cell Maturation Is Mediated by a Foxo3-mTOR Cross Talk: Outcome for Beta-Thalassemic Erythropoiesis

Abstract 176Differentiation of erythroid progenitors to mature red blood cells requires erythropoietin receptor (EpoR) signaling. Stimulation of EpoR results in Jak2-mediated activation of mainly AKT, ERK/MAPK and STAT5 signaling pathways. Although alteration of these pathways is involved with the pathophysiology of major erythroid disorders such as beta-thalassemia mechanisms by which these signals impact transcriptional programs of erythroid cell maturation are largely unknown. We have shown previously that AKT signaling is required for Epo-mediated erythroid cell maturation and identified Foxo3 transcription factor, that is negatively regulated by AKT, as a critical regulator of erythroid cell cycle, maturation and lifespan mostly through the control of oxidative stress (Marinkovic et al., JCI, 2007). In addition to Foxo3, AKT regulates several proteins including the mammalian target of rapamycin (mTOR). Here we asked how Foxo3 regulation of oxidative stress impacts erythroid cell maturation. We found that AKT/mTOR signaling pathway is constitutively activated, possibly as part of a feedback loop, in primary Foxo3−/− erythroid precursors. In addition, Epo stimulation of primary Foxo3−/− erythroid precursors led to hyperphosphorylation of Jak2, AKT, mTOR and its target p70S6 Kinase (S6K) as compared to control cells. Since Foxo3 controls levels of reactive oxygen species (ROS) in erythroid cells, and ROS are known to modify signaling proteins, we asked whether ROS are involved in the hyperactivation of AKT/mTOR signaling pathway in Foxo3−/− erythroid precursors. Combined in vivo and in vitro treatment of Foxo3−/− erythroid precursors with ROS scavenger N-Acetyl-Cysteine (NAC) reduced significantly the hyper-phosphorylation of AKT, mTOR and S6K in response to Epo. These results strongly suggest that ROS mediate the hyperactivation of AKT/mTOR signaling pathway in Foxo3−/− erythroid precursors. Next we addressed whether the imbalanced production versus maturation of Foxo3−/− erythroid precursors (Marinkovic et al., JCI, 2007) is due to the constitutive activation of AKT/mTOR signaling. This was indeed the case since in vivo treatment of Foxo3−/− mice for three weeks with the mTOR inhibitor rapamycin shifted the balance from immature towards mature erythroid cells. Interestingly while rapamycin treatment decreased cycling of Foxo3−/− erythroid progenitors as anticipated, it resulted in highly increased proliferation of Foxo3−/− mature erythroblasts as analyzed by in vivo BrdU assay. Importantly, the described Foxo3−/− erythroid phenotype was maintained on two distinct genetic backgrounds (C57BL/6 and BALB/c) in mice. These results strongly suggest that the oxidative stress-induced activation of mTOR signaling pathway mediates the imbalanced production of mature erythroid cells in Foxo3−/− mice. Given that both oxidative stress and delayed erythroid cell differentiation as seen in Foxo3−/−erythroid precursors, contribute significantly to beta-thalassemia, we asked whether the mTOR signaling is involved in the pathogenesis of this disease. Rapamycin treatment improved erythroid cell maturation in the bone marrow as analyzed by cell size, CD44, TER 119 and CD71 surface markers, and resulted in significant increase in total peripheral blood red cells and hemoglobin (1 to 1.5 g/dl increase), significant reduction in reticulocyte production as well as decrease in the spleen size of beta-thalassemic intermedia (th3/+) mice similar to what was seen in Foxo3−/− mice. Collectively these results indicate an important function for the Foxo3-mTOR cross talk in the regulation of erythroid cell maturation and suggest that rapamycin may be considered for treatment of beta-thalassemia. Disclosures:No relevant conflicts of interest to declare.

In mixed hematopoietic chimerism, the donor red cells win

In mixed hematopoietic chimerism, the donor red cells win

Differential Expression of 2 Familial Parkinson's Disease Transcripts (DJ-1 and SNCA) in Normal and Oxidant Stressed Erythroid Precursors.

Abstract 3010Poster Board II-986Superoxide dismutase 2 (Sod2) is an intramitochondrial antioxidant protein. Sod2 deficiency causes sideroblastic anemia in a murine model. Microarray analysis comparing gene transcription in normal and Sod2 deficient erythroblasts identified two differentially expressed transcripts encoding genes/proteins implicated in familial forms of Parkinson's disease—DJ-1 and alpha synuclein (SNCA). Of interest, a recent epidemiologic study drew an unexpected parallel between Parkinson's disease and anemia of unknown etiology in the elderly (1). Using qPCR and western blotting we have verified that SNCA is expressed approximately 3-fold higher in Sod2 deficient erythroid cells compared to normal cells, while DJ-1 shows a reduction in expression of 1.5 to 2-fold. Expression was examined in murine erythroleukemia cells (MEL) at baseline and following induction of differentiation with DMSO. Both SNCA and DJ-1 are unregulated upon induction of differentiation with SNCA levels gradually increasing up to 5 days post induction. DJ-1 levels peak at Day 3 after induction, with a return toward baseline levels by Day 5. Induction of SNCA and DJ-1 during erythroid differentiation suggests that these genes may be under control of erythroid specific transcription factors. Analysis of the promoter regions of both genes reveals several potential GATA binding sites. Luciferase promoter reporter constructs for both DJ-1 and SNCA were transfected into MEL cells in order to map promoter elements responsible for constitutive and differentiation induced expression, and to determine whether transcript levels were affected by increased oxidant stress in order to model the phenotype of Sod2 deficiency. DJ-1 knockout mice were examined for hematologic phenotypes including abnormalities in complete blood count, peripheral smear, marrow progenitor function in colony assays, red cell lifespan, measurement of reactive oxygen species production and oxidative damage in peripheral red cells. No differences were observed comparing DJ-1 knockout and WT controls. Crosses between DJ-1 and Sod2 mutant mice are ongoing, and we have recently transplanted compound knockout fetal liver cells in order to evaluate their ability to reconstitute irradiated host animals. The significance of differential expression of DJ-1 and SNCA in normal versus Sod2 deficient erythroid cells remains uncertain. Cell culture experiments indicate that DJ-1 plays a protective role in response to oxidative stress. Reduced expression of DJ-1 in Sod2 deficient cells may therefore exacerbate oxidative damage. Increased SNCA expression in Sod2 deficient cells may also potentiate oxidative damage—as SNCA over expression sensitizes neuronal cells to oxidant-induced death. References Cited 1. Ferrucci L, Guralnik JM, Bandinelli S, et al. Unexplained anaemia in older persons is characterised by low erythropoietin and low levels of pro-inflammatory markers. Br J Haematol. 2007;136:849-855. Disclosures:No relevant conflicts of interest to declare.

Cells Lacking the PolQ Polymerase Are Moderately Sensitive to Ionizing Radiation and the Oxidant Induced Toxicity of Paraquat and Bleomycin.

The DNA polymerase POLQ (pol theta) can bypass abasic sites in DNA and thymine glycol, a common product of reactive oxygen species damage to DNA. POLQ may also have a back-up role in base excision repair. To explore the role of POLQ in tolerance of DNA damage following irradiation, and exposure to agents that induce oxidative stress we established bone marrow stromal cell lines from the PolQ+/+ and PolQ−/− mice. Irradiation survival curves were obtained for PolQ +/+, and 2 clones of PolQ −/− cells. Dose-response experiments using the free radical generators paraquat, hydrogen peroxide and bleomycin were done on 2 clones each of PolQ +/+ and PolQ −/− cells. There was no significant difference over 23 weeks in cumulative granulopoiesis in LTBMCs from PolQ +/+ and PolQ −/− mice. PolQ+/+ cells and 2 independent clones of PolQ−/− cells were exposed to 0 – 8 Gy of gamma radiation, and colony forming ability was measured. PolQ −/− cells were more sensitive to irradiation as shown by decreased Do from 1.38 + 0.06 Gy for PolQ +/+ cells compared to 1.27 + 0.16 and 0.98 + 0.10 (p = 0.0316) Gy for PolQ −/− clones 1 and 3 respectively. Micronucleated reticulocytes were measured in peripheral blood of PolQ+/+, +/− or −/− mice after irradiation with 75 cGy or 700 cGy. Before irradiation, PolQ−/− mice had a higher fraction of MN- reticulocytes compared to PolQ+/+. Forty hr after irradiation, MN reticulocytes in PolQ−/− mice increased to 8.53 ± 1.48%, compared to 2.35 ± 0.12% and 2.55 ± 0.17% in PolQ+/+ mice (p = 0.0032). PolQ +/+ and PolQ −/− cells were exposed to paraquat (0–20 mM), hydrogen peroxide (0–40 uM) and bleomycin (0–5 ug/ml) for 1 hr prior to plating. After 7 days, the cells were stained and colonies of 50 cells or greater were counted. Statistical analysis was done using ANCOVA. PolQ −/− cells were modestly more sensitive to paraquat (p< 0.0001) and bleomycin (p< 0.0001) than PolQ +/+ cells. When the cells were treated with hydrogen peroxide, there was no significant difference in surviving fraction (p=0.7327) between the PolQ +/+ and the −/− cells. The moderate but significant sensitivity of POLQ-defective cells to bleomycin, ionizing radiation and paraquat supports the hypothesis that POLQ normally participates in helping cells tolerate DNA damage. The increased frequencies of spontaneous and radiation-induced micronuclei in peripheral blood red cells indicate that POLQ has a role in maintaining genomic integrity via limiting double-strand DNA breaks.

Effect of Licorice Root on Peripheral Blood Indexes upon Vibration Exposure

We studied the effect of continuous vibration and treatment with licorice root (Glycyrrhiza glabra L.) on peripheral blood red cells in rabbits. Active substances of licorice root accelerated metabolism in cells of the bone marrow erythroid stem, enhanced compensatory reserve of the organism, and increased animal's resistance to stress.

Decreased expression of membrane alpha4beta1, alpha5beta1 integrins and transferrin receptor on erythroblasts in splenectomized patients with beta-thalassemia intermedia. Parallel assessment of serum soluble transferrin receptors levels.

Dysfunction of cell membrane is a recognized consequence of the pathogenetic process underlying the beta-thalassemia syndromes and it is reasonable to hypothesize that surface structures crucial for the development of erythroid lineage may also be affected. The study included six adult splenectomized patients with beta-thalassemia intermedia. Expression of alpha4beta1 integrin (CD49d/CD29), alpha5beta1 integrin (CD49e/CD29) and transferrin receptor (CD71) on peripheral blood and bone marrow erythroblasts and on erythroid precursors grown in vitro was studied by flow cytometry and immunocytochemistry. Serum soluble transferrin receptor levels (sCD71) were also measured with enzyme-linked immunosorbent assay. In beta-thalassemic patients, significant reduction of CD49d, CD29 and CD71 expression was found in peripheral blood nucleated red cells, compared to patients presenting with erythroblasts in the circulation because of other diseases. Marrow erythroblasts were also deficient for the same molecules against the erythroblasts in iron deficiency anemia. All molecules tested were greatly diminished on erythroid precursors developed in vitro from the patients' cells. Serum sCD71 levels were much higher in thalassemic patients in comparison to both patients with iron deficiency anemia and healthy individuals. The loss of certain integrins and CD71 from erythroid precursors in beta-thalassemia intermedia could be attributed to a generalized membrane dysfunction, perhaps affecting the integrity of their transmembrane domains. The elevation of serum sCD71 levels may be the result of the increased red cell lineage turnover or, alternatively, may indicate increased shedding from the cells to prevent iron overload. In any case, further molecular study of the membrane components is warranted to provide a better understanding of the pathogenetic process in beta-thalassemia syndromes.

Functional requirements for phenotypic correction of murine β-thalassemia: implications for human gene therapy

AbstractAs initial human gene therapy trials for β-thalassemia are contemplated, 2 critical questions important to trial design and planning have emerged. First, what proportion of genetically corrected hematopoietic stem cells (HSCs) will be needed to achieve a therapeutic benefit? Second, what level of expression of a transferred globin gene will be required to improve β-thalassemic erythropoiesis? These questions were directly addressed by means of a murine model of severe β-thalassemia. Generation of β-thalassemic mice chimeric for a minority proportion of genetically normal HSCs demonstrated that normal HSC chimerism levels as low as 10% to 20% resulted in significant increases in hemoglobin (Hb) level and diminished extramedullary erythropoiesis. A large majority of the peripheral red cells in these mice were derived from the small minority of normal HSCs. In a separate set of independent experiments, β-thalassemic mice were bred with transgenic mice that expressed different levels of human globins. Human γ-globin messenger RNA (mRNA) expression at 7% of the level of total endogenous α-globin mRNA in thalassemic erythroid cells resulted in improved red cell morphology, a greater than 2-g/dL increase in Hb, and diminished reticulocytosis and extramedullary erythropoiesis. Furthermore, γ-globin mRNA expression at 13% resulted in a 3-g/dL increase in Hb and nearly complete correction of red cell morphology and other indices of inefficient erythropoiesis. These data indicate that a significant therapeutic benefit could be achieved with expression of a transferred globin gene at about 15% of the level of total α-globin mRNA in patients with severe β-thalassemia in whom 20% of erythroid precursors express the vector genome.

An Unusual Association of Monoclonal Gammopathy, Paroxysmal Nocturnal Haemoglobinuria and Myelodysplastic Syndrome Transformed into Acute Myeloid Leukaemia: Coexistence of Triple Clonal Disorders

An unusual association of paroxysmal nocturnal haemoglobinuria (PNH), myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML) and monoclonal gammopathy is reported. A 60–year old male, who had a history of IgA monoclonal gammopathy, presented with haemoglobinuria and colic pain. Flow cytometry showed CD55negative/59dim peripheral red cells, and bone marrow examination disclosed MDS. Eleven months, he developed later AML with disappearance of the PNH clones, although the monoclonal gammopathy persisted. The relationship between PNH and MDS has not fully been assessed, although our findings indicate that these triple clonal disorders, all coexisted in one patient.

Patterns of organ-specific engraftment by stem cell subsets and committed progenitors.

The kinetics of blood and organ engraftment following transplants of defined populations of hematopoietic stem/progenitor cells were investigated utilizing cell populations defined by surface antigen and rhodamine-123 staining. While long-term repopulating stem cells, short-term multipotent progenitors and committed progenitors all reconstituted peripheral blood red cells and splenic cellularity, only the population of cells that includes highly enriched long-term repopulating stem cells (Thy-1.1lowLinnegSca-1+Rh123low) reconstituted marrow cellularity. In addition, peripheral blood platelet and nucleated cell count increased only after transplant of the long-term repopulating population. These results argue that the major cell population that functions to reconstitute hematopoiesis after bone marrow transplantation is a primitive, marrow-homing stem cell. Transplantation of highly enriched multipotent progenitors that lack long-term reconstituting potential had no impact on hematopoietic recovery, apart from a transient increase in circulating erythrocytes. These results suggest that the primary cell population that functions to reconstitute hematopoiesis in a transplant setting is the long-term repopulating stem cell. This observation is discussed in the context of the normal hematopoietic process.

Amifostine Plus Granulocyte Colony-stimulating Factor Therapy Enhances Recovery from Supralethal Radiation Exposures: Preclinical Experience in Animal Models

A murine model was used to explore whether the cytoprotective agent amifostine (WR-2721) can be used to protect a critical fraction of haemopoietic stem cells against radiation, and whether granulocyte colony-stimulating factor (G-CSF) can then be used to stimulate the protected cells to proliferate and reconstitute the haematopoietic system. Groups of C3H/HeN mice treated with 200 mg/kg amifostine i.p. 30 min before 60Co irradiation and/or 125 micrograms/kg G-CSF subcutaneously from days 1-16 post irradiation were compared. The dose reduction factor (DRF) of the combination of amifostine and G-CSF from LD50/30 values was greater than the sum of the DRFs for amifostine and G-CSF individually. Acceleration of recovery bone marrow and splenic multipotent stem cells (CFU-s) and granulocyte-macrophage progenitor cells (GM-CFC), as well as of peripheral blood red and white cells and platelets, was greatest in mice treated with amifostine plus G-CSF. These studies suggest that amifostine and recombinant haematopoietic growth factors can be used in combination to reduce myelosuppression and lethality associated with radiation or radiomimetic drugs