Number Of Red Cells Research Articles

A simple test for the cleavage activity of customized endonucleases in plants.

BackgroundAlthough customized endonucleases [transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs)] are known to be effective agents of mutagenesis in various host plants, newly designed endonuclease constructs require some pre-validation with respect to functionality before investing in the creation of stable transgenic plants.ResultsA simple, biolistics-based leaf epidermis transient expression test has been developed, based on reconstituting the translational reading frame of a mutated, non-functional yfp reporter gene. Quantification of mutation efficacy was made possible by co-bombarding the explant with a constitutive mCherry expression cassette, thereby allowing the ratio between the number of red and yellow fluorescing cells to serve as a metric for mutation efficiency. Challenging either stable mutant alleles of a compromised version of gfp in tobacco and barley or the barley MLO gene with TALENs/RGENs confirmed the capacity to induce site-directed mutations.ConclusionsA convenient procedure to assay the cleavage activity of customized endonucleases has been established. The system is independent of the endonuclease platform and operates in both di- and monocotyledonous hosts. It not only enables the validation of a TALEN/RGEN’s functionality prior to the creation of stable mutants, but also serves as a suitable tool to optimize the design of endonuclease constructs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0118-6) contains supplementary material, which is available to authorized users.

Fractionated stem cell infusions for patients with plasma cell myeloma undergoing autologous hematopoietic cell transplantation

We conducted a phase II trial investigating the impact of fractionated hematopoietic cell infusions on engraftment kinetics and symptom burden in patients with plasma cell myeloma (PCM) undergoing autologous hematopoietic cell transplant (AHCT). We hypothesized that multiple hematopoietic cell infusions would reduce duration of neutropenia and enhance immune recovery resulting in a better tolerated procedure. Twenty-six patients received high-dose melphalan followed by multiple cell infusions (Days 0, +2, +4, +6) and were compared to PCM patients (N = 77) who received high-dose melphalan and a single infusion (Day 0) (concurrent control group). The primary endpoint was number of days with ANC <500K/mcL. Symptom burden was assessed using the MSK-modified MD Anderson Symptom Inventory. Median duration of neutropenia was similar in study (4 days, range 3–5) and control patients (4 days, range 3–9) (p = 0.654). There was no significant difference in the number of red cell or platelet transfusions, days of fever, diarrhea, antibiotics, number of documented infections, or length of admission. Symptom burden surveys showed that AHCT was well-tolerated in both study and control patients. We conclude that fractionated stem cell infusions following high-dose melphalan do not enhance engraftment kinetics or significantly alter patients’ clinical course following AHCT in PCM.

The Zebrafish Provides Mechanistic Insights into the Role of Poly(A)-Specific Ribonuclease (PARN) in Hematopoietic Stem Cell (HSC) Homeostasis and Bone Marrow Failure

Development of tissues during embryogenesis and their homeostasis after formation are highly regulated by expression of coding and non-coding RNAs. Deadenylation is a core mechanism that regulates RNA function and fate by controlling turnover, abundance and maturation of RNA. Factors that promote or inhibit deadenylation control hematopoietic stem cell (HSC) homeostasis, and inhibition of deadenylation limits differentiation of the HSCs. Importantly, RNA biogenesis has emerged as a mechanism underlying several inherited bone marrow failure syndromes (IBMFSs), such as Diamond Blackfan anemia, dyskeratosis congenita (DC) and Shwachman-Diamond syndrome. Poly(A)-specific ribonuclease (PARN) is a major deadenylation factor and demonstrates high specificity for single-stranded poly (A) tails of various RNA species. We recently identified biallelic mutations in PARN as a cause of hematopoietic failure and profound hypomyelination, similar to the severe form of DC, Hoyeraal-Hreidersson syndrome.We developed a zebrafish model to characterize the hematopoietic phenotype of a patient identified to have severe inherited bone marrow failure resulting from a combined deletion of PARN on one allele and missense mutation in the other. Zebrafish posses a single parn ortholog. Zebrafish parn protein shares homology and high sequence identity (~64%) to its human counterpart. Embryos were injected with either translation start-site or splice-site-blocking morpholino at the one-cell stage. Both morpholino injections resulted in anemic embryos at 48 hours post fertilization (hpf), as evidenced by reduced o-dianisidine staining and gata1 expression by whole-mount in situ hybrization and GFP+ red cell numbers by fluorescence-activated cell sorting (FACS). Morphant embryos also demonstrated reduced expression of myeloid cell markers including l-plastin, myeloperoxidase, and macrophage expressed gene 1 and were leukopenic as evidenced by reduced number of GFP+ myeloid cells. FACS analysis revealed that fluorescently labeled HSCs were increased in parn morphants. Early hematopoietic markers, lmo2 and fli1, expressed in hemogenic and vascular tissue respectively, were also overexpressed in parn morphants. Furthermore, there was reduced global cell proliferation in morphant embryos as determined by phosphohistone H3 antibody staining. These findings suggest that the absence of parn results in a developmental arrest at the HSC stage with an inability to differentiate into leukocyte or erythroid lineages. Similarly, human cell culture data from PARN-deficient HSC/progenitor cells demonstrated markedly reduced colony forming capacity. By modeling parn deficiency in the zebrafish, we validate for the first time an IBMFS that results from biallelic mutations in a major deadenylating protein. Moreover, our zebrafish studies provide insight into the role of parn in maintaining HSC homeostasis/differentiation as the origin of the pancytopenia observed in this patient. Permanent knockouts in the zebrafish using CRISPR/Cas9 technology are underway, which will enable tracking the hematopoietic phenotype into adulthood. These studies have set the stage for critical translational research in a rare form of bone marrow failure as well as new insight into HSC regulation. DisclosuresNo relevant conflicts of interest to declare.

Lack of Beta-1 Integrin Limits Stress Erythropoiesis and Splenomegaly in Beta-Thalassemia

After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β−thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interrupting the interaction between macrophages and erythroblasts (MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. We speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta−1 (Itgβ1) and also intracellular molecules such as focal adhesion kinase (Fak1), Talin−1 and Sharpin might play a role in stress erythropoiesis. Furthermore, there is increased interaction between Itgb1 and Fak1 in erythroblasts co−cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgβ1 or Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgβ1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. Enucleation of erythroblasts was accompanied by a marked loss of Itgβ1 expression, indicating that Itgβ1 may be involved in erythroblast enucleation and differentiation. We crossed Hbbth3/+ mice with animals in which Itgβ1 or Fak1 were floxed and carrying an inducible Cre−recombinase (Mx1−Cre). From these animals, we investigated three different models; two obtained from breeding (Hbbth3/+−Itgβ1fl/fl−Mx1−Cre and Hbbth3/+−Fak1fl/fl−Mx1−Cre) and one by bone marrow transplant (BMT) of hematopoietic stem cells (HSCs) of Hbbth3/+−Itgβ1fl/fl −Mx1−Cre animals into wt mice to generate thalassemic animals that expressed the floxed Itgβ1 only in hematopoietic cells. After serial administration of Poly(I)−Poly(C) [poly(I:C)] the animals were analyzed for their erythropoiesis in the bone marrow and spleen. All the animals treated with poly(I:C) showed populations of Itgβ1 or Fak1 negative cells in the bone marrow and spleen. This indicated that all the HSCs were successfully depleted of the Itgβ1 or Fak1 gene. Interestingly, the spleen weight of all the treated animals was reduced, on average, 50% compared to untreated thalassemic mice. Similar results were seen also in Hbbth3/+−Itgβ1fl/fl−Mx1−Cre animals generated through BMT. Therefore, Itgβ1 and Fak1 might contribute to the pathophysiology of thalassemia and their removal might result in reduced stress erythropoiesis, erythroid proliferation and, as a consequence, amelioration of splenomegaly. Iron analysis and quantification of Erythroferrone (ERFE) are in progress to evaluate the impact of depleting Itgβ1 and Fak1 on these mechanisms. We are now in the process of identifying compounds that target MEI and, in particular, Itgβ1. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. DisclosuresFeldman:Bayer ealthCare Phamaceuticals Inc.: Employment. Rivella:isis Pharmaceuticals: Consultancy; Merganser Biotech: Other: Stock options; Novartis Pharmaceuticals: Consultancy; Medgenics Pharmaceuticals: Consultancy; Bayer Healthcare: Consultancy, Research Funding.

Hemolytic vascular inflammation: an update.

Intravascular hemolysis can occur in a number of diseases,including sepsis, malaria and the hemolytic anemias [sicklecell disease (SCD), paroxysmal nocturnal hemoglobinuria,etc.], as well as following certain events such as mismatchedtransfusions. The pathophysiological significance of intravas-cular hemolysis had been largely ignored in diseases suchas SCD save for its effect on red cell numbers, however, rel-atively recent studies have shed light on the major effectsthat hemolyticprocesseshaveonvascularbiology,particularlyon the endothelium, inflammatory processes and oxidativestress.Under normal circumstances, hemoglobin (Hb) is con-tained in the red blood cells (RBCs), and employed primarilyfor oxygen transport. However, the destruction of RBC (i.e.hemolysis) results in the release of Hb into the circulation.If this hemoglobin is not neutralized immediately into lesstoxic metabolites by specialized scavenger proteins, such ashaptoglobin and hemopexin, as can occur when extensivehemolysis overwhelms innate protective mechanisms, thiscell-free Hb causes chaos.

Physiological and thermographic response to heat stress in zebu cattle

The objective of this study was to evaluate the heat tolerance of five zebu breeds using physical, physiological and hematological traits as well as thermographic responses. Forty cows of the Gir, Girolando, Nelore, Sindhi and Indubrasil breeds (eight cows each), approximately three years of age, were evaluated. Body weight, withers and hump heights as well as thoracic circumference were recorded. The density and length of the hair was obtained by collecting one square centimeter in the rump region and skin color using the CIELAB system. Rectal temperature, heart and respiratory rates were evaluated during the morning at 4:30h, and in the afternoon, at 14:30h, with six repetitions. Blood samples were collected for hematological evaluation. The surface temperature was obtained using an infrared camera FLIR® T400. Two images were taken from each animal, one laterally of the whole body and the other of the head region. Air temperature, wind speed, relative humidity were obtained from a mobile weather station. The statistics analysis included an analyzes of variance, principal factors, as well as cluster, discriminant and canonical analyzes, logistic regression and calculation of odds ratio. There were significant differences in the rectal temperature, heart and respiratory rates between breeds. Gir and Indubrasil breeds had the highest rectal temperatures. Breed was significant for surface temperatures and showed that physical and physiological factors affected breeds in different ways. Eye and brain surface temperatures were the most affected by environmental parameters. Also, environmental parameters affected packed cell volume and red cell number. Odds ratio test showed that the Gir breed was three times more likely to have higher rectal temperature compared with Sindhi as confirmed by the logistic regression. When the black globe temperature approached 35°C, the probability of the Gir animals having rectal temperatures above normal was approximately 70%. Gir was the breed least adapted to climate conditions of the experiment while the Sindhi and Girolando breeds showed the best physiological response to thermal stress.

Long-term effects of growth hormone (GH) replacement therapy on hematopoiesis in a large cohort of children with GH deficiency.

The aim of our prospective case-control study was to evaluate long-term effects of GH replacement therapy on erythrocytes parameters, leukocytes, and platelets numbers in a large cohort of children with isolated GH deficiency (GHD). Hemoglobin (Hb) concentration, hematocrit (Hct), mean corpuscular volume, mean corpuscular hemoglobin, red cell distribution width, number of erythrocytes, leukocytes, neutrophils, lymphocytes, monocytes and platelets, ferritin, and C-reactive protein were evaluated in 85 children with isolated GHD (10.20±3.50years) before and annually during the first 5years of GH replacement therapy and in 85 healthy children age and sex comparable to patients during 5years of follow-up. Compared with controls, GHD children at study entry showed lower Hb (-1.18±0.87 vs. -0.40±0.90 SDS, p<0.0001), red cells number (-0.24±0.81 vs. 0.25±1.14 SDS, p<0.0001), and Hct (-1.18±0.86 vs. -0.68±0.99 SDS, p<0.0001). Twelve GHD patients (14%) showed a normocytic anemia. GH therapy was associated with a significant increase in Hb, Hct, and red cells number which became all comparable to controls within the first 2years of treatment. Moreover, hemoglobin levels normalized in all anemic GHD patients after 5years of therapy. No difference between patients and controls was found in leukocytes and platelets numbers neither at baseline nor during the study. GHD in childhood is associated with an impairment of erythropoiesis which causes a normocytic anemia in a considerable percentage of patients. GH replacement therapy exerts a beneficial effect leading to a significant increase of erythrocytes parameters and recovery from anemia. Neither GHD nor GH replacement treatment exerts effects on leukocytes or platelets numbers.

Effects of dietary peppermint (Mentha piperita) on growth performance, chemical body composition and hematological and immune parameters of fry Caspian white fish (Rutilus frisii kutum).

Peppermint (Mentha piperita L.) is a very popular herb. While numerous effects have been described in mammals, its effects on fish have received so far limited attention. The effects of dietary administration of peppermint on fry Caspian white fish (Rutilus frisii kutum) were studied. Fish were divided into 4 groups before being fed diets supplemented with 0% (control), 1%, 2% and 3% of peppermint extracts for 8 weeks. Dose-dependent increases of growth parameters (WG and SGR), mucus skin (protein concentration, alkaline phosphatase and antimicrobial activity) and seric (lysozyme and IgM) and blood leucocyte respiratory burst activities and different hematological parameters (number of red and white cells, seric hemoglobin and hematocrit content) were recorded in fry fish fed supplemented diets. However, the dietary peppermint supplements have different effects on the number of blood leucocytes depending on the leucocyte cell type. While no significant differences were observed in the number of blood monocytes and eosinophils, the number of neutrophils and lymphocytes was increased and decreased, respectively, on fish fed peppermint enriched diets, respect to the values found in control fish. Present results corroborate that dietary administration of peppermint promotes growth performance and increases the main hematological and immune humoral (both mucosal and systemic) parameters of fry Caspian white fish. This study may provide new applications of peppermint and, at the same time, promote rational development and utilization of peppermint resources.

Discrimination of various thalassemia syndromes and iron deficiency and utilization of reticulocyte measurements in monitoring response to iron therapy.

Decreased hemoglobinization of red cells resulting in hypochromia and microcytosis are the main features of thalassemia syndromes, and also of iron deficiency anemia (IDA). A simple and reliable method is required to distinguish the two conditions in the routine laboratories. In this study we analyzed the red cell and reticulocyte parameters from 414 samples of various types of thalassemias and IDA and discovered a variety of discriminating criteria including a discrimination index (DI) which should be useful for differential diagnosis. Slightly decreased MCV and CH are suggestive of α-thalassemia 2, Hb CS, and Hb E heterozygotes whereas the increased Rbc counts are obvious in α-thalassemia 1 and β-thalassemia. In Hb E, the number of microcytic red cells was greater than the number of hypochromic red cells resulting in an increased M/H ratio. Hb H diseases are characterized by a higher number of hypochromic red cells and decreased CHCM, while broadening of hemoglobin concentration histogram results in increased HDW in β-thalassemia diseases. Iron deficiency anemia results in hypochromic-microcytic red cells and increased RDW. The number of reticulocyte with %High Retic and CHr value were increased in the first month of iron supplementation indicating the response to iron therapy.

Preclinical Determination of the Efficacy of Epigenetic Therapy in High Risk Myeloid Disease Using the Zebrafish Model

Epigenetic therapy implies the use of drugs that target regulators of gene expression, such as DNA and histone methylation or histone acetylation, without altering the DNA coding sequence. Epigenetic mechanisms have been found to be perturbed in myeloid diseases, and in fact, the DNA demethylating agents, 5-azacytadine and decitabine are FDA approved drugs for myelodysplastic syndrome. However, use of epigenetic therapies in other types of myeloid disease including acute myeloid leukemia (AML) has been met with variable success, suggesting efficacy might be improved by preselecting subtypes of disease in which there is a high degree of epigenetic dysregulation. AML has recently been found to be associated with a host of epigenetic abnormalities including mutations in DNA methyltransferase 3A (DNMT3A), ten-eleven translocation-2 (TET2) and enhancer of zeste homolog 2 (EZH2).We generated a transgenic zebrafish model of high risk myeloid disease expressing the human NUP98-HOXA9 (NHA9) fusion oncogene, a genetic lesion for which epigenetic dysregulation has not previously been identified. Transgenic embryos exhibit an increase in immature myeloid cells at the expense of erythroid cells and adult fish develop a myeloproliferative neoplasm (MPN). Leveraging this model in a microarray screen, we identified 3-fold elevated levels of the epigenetic regulator, dnmt1, the major maintenance methyltransferase, for the first time in high risk AML. Decitabine specifically inhibits DNMT1 and treatment of NHA9 transgenic embryos with 75µM decitabine restored normal hematopoiesis, as evidenced by normal numbers of leukocytes and red cells. Moreover, using hematopoietic stem cell (HSC) reporter lines and whole mount in situ hybridization, we identified a 2-3 fold increase in this population in NHA9 embryos, suggesting the HSC as the cell of origin in this disease. Interestingly, 75µM decitabine therapy also restored normal HSC numbers. Strikingly, we discovered synergy when we combined sub-monotherapeutic doses of DNMT1 inhibitors, decitabine (10-25µM) or zebularine (100µM), plus histone deacetylase inhibitors, valproic acid (25-100µM) or trichostatin A (250nM), to inhibit the effects of NHA9 on hematopoiesis.To determine if NHA9 expression directly results in changes to DNA methylation, we performed MeDIP-Seq on genomic DNA from pools of untreated and decitabine-treated NHA9 or control embryos. Untreated NHA9 embryos displayed significantly higher methylation levels at the regions of gene promoters compared to control embryos, which were restored to control levels following treatment with 75µM decitabine. Similarly, combination therapy with 10µM decitabine and 25µM valproic acid significantly reduced methylation to near control levels following a 5 hour treatment, while prolonged exposures to these same doses resulted in profound global hypomethylation.These findings elucidate underlying mechanisms in the pathogenesis of NHA9-induced myeloid disease and propose novel actionable epigenetic drug targets. Furthermore, we highlight the opportunities inherent in the zebrafish model as a unique in vivo platform for the preclinical screening of epigenetic based combination therapy. DisclosuresNo relevant conflicts of interest to declare.

Beta-1 Integrin Controls Homing and Expansion of Erythroid Cells in Stress Erythropoiesis and ß-Thalassemia

After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β-thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes.Interupting the interaction between macrophages and erythroblasts (macrophage-erythroblast interaction, MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. Targeting MEI, using a number of approaches, caused a significant improvement in blood parameters in β-thalassemia intermedia (BTI) mouse models (Hbbth3/+) and a rapid and dramatic improvement in splenomegaly, an outcome that is relevant for clinical practice. Importantly, MEI is not critical for hematopoiesis under non-stress conditions, and ablation of this interaction in normal mice showed minimal effects on blood parameters. As our initial observations indicate that MEI is essential to support stress erythropoiesis, we investigated adhesion molecules that might activate downstream pathways in erythroblasts that regulate cell proliferation. We also speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver.Our studies in erythroblasts indicate that integrin beta 1 (Itgb1) and also intracellular molecules such as Fak1, Talin1 and Sharpin might play a role in stress erythropoiesis. There is increased interaction between Itgb1 and Fak1 in erythroblasts co-cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgb1 and Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgb1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. More specifically, when measuring the content of DNA, we were able to demonstrate that enucleation of erythroblasts was accompanied by a marked loss of Itgb1 expression, indicating that there may be an important role for Itgb1 in erythroblast enucleation, and differentiation in general. Lack of Itgb1 in thalassemic mice prevents erythroid cells from homing to and expanding in the spleen, the major source of chronic stress erythopoiesis in this disorder. In particular, such a role of Itgb1 is supported by our analysis of thalassemic mice in which this molecule was partially depleted by induction of the Cre recombinase. These animals were generated by crossing th3/+ mice with animals in which Itgb1 was floxed and carrying an inducible Cre-recombinase (Mx1-CRE). We utilized the BM of these animals (Hbbth3/+, Itgb1fl/fl, Mx1-CRE) to generate thalassemic animals that expressed the floxed Itgb1 only in hematopietic cells. After serial administration of polyI:C the animals were analyzed for their erythropoiesis in the bone marrow and spleen. Interestingly, all the animals analyzed show chimeric populations of Itgb1 positive and negative erythroid cells in the bone marrow. This indicated that not all the HSCs were successfully depleted of the Itgb1 gene. However, when we investigated Itgb1 in the spleen, we observed only erythroid cells positive for the expression of this adhesion molecule. This last observation strongly suggests that depletion of Itgb1 prevents homing and expansion of erythroid cells in the spleen and drugs that by inhibit Itgb1 could reduce erythroid spleen colonization, splenomegaly and limit erythropoiesis.We are now in the process of identifying compounds that target MEI. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. DisclosuresCasu:Merganser Biotech : Research Funding; Isis Pharmaceuticals, Inc.: Research Funding.

Concurrent Treatment with Minhepcidin and Deferiprone Improves Anemia and Enhances Reduction of Spleen Iron in a Mouse Model of Non-Transfusion Dependent Thalassemia

Individuals affected by non-transfusion-dependent thalassemia (NTDT) develop severe ineffective erythropoiesis that causes a number of serious clinical morbidities, such as chronic anemia, splenomegaly and systemic iron overload requiring chelation therapy. In NTDT, low hepcidin levels caused by ineffective erythropoiesis result in increased iron absorption which may in turn exacerbate erythroid cell damage, apoptosis and ineffective erythropoiesis. Minihepcidins (MH) are short peptide mimetics that reproduce the iron restrictive effects of hepcidin. Using a mouse model of NTDT (Hbbth3/+), we previously showed that MH-induced iron restriction significantly reduced erythroid cell damage, leading to reduced ineffective erythropoiesis and improved anemia. Accumulation of tissue iron was significantly diminished in MH treated animals. In clinical settings, use of MH for the treatment of NTDT would likely be concomitant with oral chelation therapy. Therefore we conducted studies in the (Hbbth3/+) mouse to evaluate whether the concurrent use of the oral iron chelator deferiprone (DFP) and MH would alter a) the hematological benefit of MH or b) the tissue iron reduction benefit of DFP. MH may improve the efficacy of DFP by reducing dietary iron absorption, thus increasing net reduction in tissue iron.Studies were performed in six week old mice using MH M004, which reduces cell surface expression of ferroportin with an EC50of 9.7 nM and causes >80% reduction in serum iron at a dose of 7.5 mg/kg in the rat. M004 (2.65 mg/kg) or vehicle control was administered twice weekly by subcutaneous injection for six weeks. Half of the mice in each group had free access to water containing DFP (1.25 mg/mL).Treatment with MH alone produced a profile of hematological changes resulting in a significant increase in circulating hemoglobin of 1.6 g/dL. Flow cytometry studies of bone marrow erythroid populations from MH-treated animals demonstrated an increase in the relative proportion of mature erythroid cells, reduced apoptosis and reduced levels of ROS. Parameters of erythrocyte damage (red cell distribution width, red cell morphology, red cell survival time) were improved and an increase in peripheral red cell number was observed. Reticulocyte count and spleen size were both reduced, reflecting improved erythropoietic efficiency. In separate studies in Hbbth3/+ mice that also expressed a hypoxia induced luciferase-reporter gene, treatment with MH resulted in reduction of tissue hypoxia. Mice treated with DFP alone showed no hematologic improvement compared to vehicle control whereas mice treated with MH and DFP together showed similar hematologic benefit as mice treated with MH alone (Hb increase of 2.3 g/dL versus control).Both DFP and MH administered separately caused a reduction in total liver iron compared to vehicle controls but the different was only statistically significant in MH treated animals. When administered concurrently, the combination of DFP and MH caused a further non-significant reduction compared to either agent alone. Although spleen iron/g wet weight was increased by MH, reflecting the iron-sequestering effect of hepcidin, total spleen iron was not significantly increased because spleen size was also reduced. DFP was equally effective in reducing spleen iron/g wet weight with or without concurrent MH treatment. However total spleen iron was non-significantly reduced (-35%) in DPF-treated mice compared to vehicle controls (1,435 ± 627 µg (mean ± SEM) vs 2,210 ± 213 µg) but a larger, statistically significant reduction in spleen iron was observed in MH + DFP treated mice compared to MH alone (-50%, 1,258 ± 414 vs 2,531 ± 246, p<0.05).In conclusion, the beneficial hematological effects of MH treatment in this model of NTDT remained even during concurrent treatment with DFP. DFP had no hematological benefits when used as a single agent, reflecting the inability of iron chelators to cause clinically meaningful iron restriction. Reduction in tissue iron by DFP was either unaffected or enhanced by concurrent treatment with MH. The improved effect of DFP on spleen iron burden in the presence of MH may reflect the complementary effects of these agents to increase iron excretion and reduce dietary iron absorption respectively. Enhancement of the clinical efficacy of iron chelators may be an important benefit of MH therapy in addition to the observed increase in circulating hemoglobin. DisclosuresCasu:Isis Pharmaceuticals, Inc.: Employment; Merganser Biotech LLC: Employment. Nemeth:Merganser Biotech LLC: Stockholders Other. Ganz:Merganser Biotech LLC: Stockholders Other. MacDonald:Merganser Biotech LLC: Employment, Equity Ownership. Rivella:bayer: Consultancy, Research Funding; merganser Biotech LLC: Consultancy, Research Funding, Stock options, Stock options Other; isis Pharmaceuticals, Inc.: Consultancy, Research Funding.

Hypoxia-modulated gene expression profiling in sea urchin (Strongylocentrotus nudus) immune cells

Hypoxia is an issue that affects ocean coastal waters worldwide. It has severe consequences for marine organisms, including death and rapid adaptive changes in metabolic organization. Although some aquatic animals are routinely exposed and resistant to severe environmental hypoxia, others such as sea urchins (Strongylocentrotus nudus) have a limited capacity to withstand this stress. In this study, hypoxia induced a significant increase in the number of red spherule cells among coelomocytes, which function as immune cells. This suggests that sea urchin immune cells could be used as a biological indicator of hypoxic stress. In the current study, we used cDNA microarrays to investigate the differential expression patterns of hypoxia-regulated genes to better understand the molecular mechanisms underlying the response of immune cells to hypoxia. Surprisingly, the predominant major effect of hypoxia was the widespread suppression of gene expression. In particular, the expression of RNA helicase and GATA-4/5/6 was decreased significantly in response to hypoxia, even in field conditions, suggesting that they could be utilized as sensitive bioindicators of hypoxic stress in the sea urchin.

Japanese guidelines of the management of hematuria 2013.

Japanese guidelines of the management of hematuria 2013.

Conditioning out-of-date bank-stored red blood cells using a cell-saver auto-transfusion device: effects on numbers of red cells and quality of suspension fluid.

We investigated the utility of a cell-saver device for processing out-of-date red blood cells, by washing twenty bags of red blood cells that had been stored for between 36 and 55days. The volume of recovered cells, and the characteristics of the suspension fluid, were measured before and after treatment. The ratio of free haemoglobin to total haemoglobin was up to 0.02 before processing, and up to 0.011 afterwards, changing by between -0.013 and +0.003. This ratio met the current standard for free haemoglobin (less than 0.008 in more than 75% of samples), both before and after processing. Ninety-three percent of red blood cells survived the process. Potassium ion concentration fell from above 15mmol.l(-1) in all cases, to a mean of 6.4mmol.l(-1) (p<0.001). The pH rose to a mean value of 6.44 (p=0.001). Lactate ion concentration fell to a mean value of 14mmol.l(-1) (p<0.001). Sodium ion concentration rose from a mean value of 93mmol.l(-1) to a mean value of 140mmol.l(-1) (p<0.001). A useful proportion of out-of-date red blood cells remained intact after conditioning using a cell-saver, and the process lowered concentrations of potentially toxic solutes in the fluid in which they were suspended.

The impact of initial cerebrospinal fluid (CSF) findings on outcome among patients with NCI standard (SR) and high-risk (HR) B-lymphoblastic leukemia (ALL): A report from the Children’s Oncology Group (COG) Studies AALL0331 and AALL0232.

10016 Background: To determine the prognostic significance of variable numbers of white cells (WBC), blasts and/or red cells (RBC) in diagnostic CSF samples of ALL patients, COG protocols, AALL0331...

P945 CLINICAL OUTCOME OF GASTRIC VARICES IN PATIENTS WITH PORTAL HYPERTENSION WITH OR WITHOUT CIRRHOSIS WITHOUT A PREVIOUS HISTORY OF BLEEDING

Background and Aims: Gastrointestinal vascular anomalies can cause chronic anaemia, a clinical challenge in terms of endoscopic therapy, transfusion and hospital admissions. Evidence suggests Thalidomide as a therapeutic option for such patients. We aim to determine the efficacy and side-effect profile of Thalidomide in patients with gastrointestinal vascular anomalies. Methods: A retrospective case-note review 12-months preand post-Thalidomide therapy. Results: Patients on Thalidomide due to failed standard therapy between the years 2004 and 2013 were included. 17 patients comprising 7 women and 10 men with a median age of 68.5 years (range 48–87 years) were identified. Endoscopic diagnoses were; portal hypertensive gastropathy (PHG) (n =5), Gastric Antral Vascular Ectasia (GAVE) (n = 9), PHG/GAVE (n =4), upper gastrointestinal vascular malformation (GIVM) (n =5) and lower GIVM (n=2). 11 patients continued treatment beyond 3 months and 5 out of 11 took Thalidomide for atleast 12 months. There was reduction in transfusion frequency by 65% (p < 0.05), number of red cell concentrate units by 71% (p < 0.05), number of OGDs by 81% (p < 0.05), number of argon treatments by 75% (p < 0.05), and number of hospital admissions by 71% (p < 0.05) for the 11 patients. No statistically significant improvement was seen in patients who received less than 3 months of therapy. 27% developed side effects (3/11). The side effects reported were peripheral neuropathy, fatigue and pancytopenia, which lead to cessation of therapy. Conclusions: Thalidomide has a role in the management of gastrointestinal vascular lesions resistant to conventional treatment, drug side effects are common and require monitoring. P945 CLINICAL OUTCOME OF GASTRIC VARICES IN PATIENTS WITH PORTAL HYPERTENSION WITH OR WITHOUT CIRRHOSIS WITHOUT A PREVIOUS HISTORY OF BLEEDING E. Ahn, P. Kortan, G. May, N. Marcon, G. Kandel, Y.I. Kim, S. Grover, M. Cirocco, F. Wong. Division of Gastroenterology, Toronto General Hospital & University of Toronto, Division of Gastroenterology, St. Michael’s Hospital, Toronto, ON, Canada E-mail: eric.ahn@mail.utoronto.ca

A survey of O RhD‐negative red cells in the South West: stocks and distribution compared to use and wastage

O RhD-negative (ONeg) red cells can be used in an emergency for recipients of other blood groups. Matching supply and demand is currently a challenge; therefore, any service redesign, using more remote blood fridges, must consider ONeg red cell availability. To identify whether the number of fridges stocking emergency ONeg units correlates with use and wastage. The number and distribution of ONeg red cells was requested from the hospitals in South West England. For NHS Hospitals, comparison was made with ONeg National Health Service (NHS) organisation--NHS Blood and Transplant (NHSBT) issues (ONeg as a proportion of all red cells), wastage and the proportion of ONeg units given to ONeg patients (ONeg-to-ONeg use). Correlations were performed using Spearman's rank correlation coefficient. Of the 23 hospitals, 21 responded. Four hundred and forty three ONeg units were held across the region--56% as stock and the remaining as emergency units. ONeg issues increased with the number of fridges holding emergency units (ρ = 0.48, significance 0.046). No correlation was found between the number of fridges and ONeg wastage or ONeg-to-ONeg use. A longer unit shelf life on rotation back to stock was associated with lower wastage (ρ = -0.597, significance 0.009). Although there was a weak correlation between fridge numbers and overall percentage ONeg use, there was no correlation with ONeg wastage.

Hematology analyzer for cell counting of hemorrhagic cerebrospinal fluid

Objective To explore the feasibility of hematology analyzer for the cell counting of hemorrhagic cerebrospinal fluid.Methods 133 hemorrhagic cerebrospinal fluid specimens were collected.Traditional microscope counting was used in the control group and ABX-pentra-60 hematology analyzer was used in the observation group to count the red cells.The data were analyzed using paired t-test statistical analysis.Results The number of red cells was 916.72 × 109/L in the control group and was 869.66 × 109/L in the observation group,wihtout statistical difference (t=1.091,V=132,P=0.277 [bilateral]).Conclusion ABX-pentra-60 hematology analyzer can be use in the cell counting of hemorrhagic cerebrospinal fluid. Key words: Hemorrhagic cerebrospinal fluid; Cell counting; Hematology analyzer; Analysis

Relación entre células sanguíneas y variables metabólicas en mujeres indígenas de diferentes edades que viven a gran altitud

Some studies have found a positive relationship between the concentration of blood cells and metabolic alterations, suggesting that the concentration of blood cells should be included as a component of metabolic syndrome. However, these studies have been done at a low altitude, and it is unknown if this relationship is maintained at a high altitude, where the low atmospheric pressure and the high hypoxia affect the number of red cells and the presence of metabolic alterations. The aim of the present study was to determine the relationship between red blood cells and metabolic variables in Otomi women from Ixtenco-Tlaxcala living at 2500 meters above sea level. METHODS. The body mass index (BMI), concentration of total cholesterol, triglycerides, and complete blood count were measured in 309 women. Data were analyzed through correlations and binary logistic regressions. RESULTS.The BMI was positively correlated with the concentration of erythrocytes and hemoglobin, as well as with hematocrit. Independently of the age, women with overweight/obesity and dislipidemias have a big risk to have erythrocitosis, and low risk to have anemia. DISCUSSION. The relationship between the concentration of blood cells and obesity/ dislipidemias is affected by the altitude.