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ARHGAP21 is a Rho-GAP that controls GTPases activity in several tissues, but its role on liver lipid metabolism is unknown. Thus, to achieve the Rho-GAP role in the liver, control and ARHGAP21-haplodeficient mice were fed chow (Ctl and Het) or high-fat diet (Ctl-HFD and Het-HFD) for 12 weeks, and pyruvate and insulin tolerance tests, insulin signaling, liver glycogen and triglycerides content, gene and protein expression, and very-low-density lipoprotein secretion were measured. Het mice displayed reduced body weight and plasma triglycerides levels, and increased liver insulin signaling. Reduced gluconeogenesis and increased glycogen content were observed in Het-HFD mice. Gene and protein expression of microsomal triglyceride transfer protein were reduced in both Het mice, while the lipogenic genes SREBP-1c and ACC were increased. ARHGAP21 knockdown resulted in hepatic steatosis through increased hepatic lipogenesis activity coupled with decreases in CPT1a expression and very-low-density lipoprotein export. In conclusion, liver of ARHGAP21-haplodeficient mice are more insulin sensitive, associated with higher lipid synthesis and lower lipid export.

NR4A3 is a key tumor suppressor in myeloid malignancy, mice lacking both NR4A1 and family member NR4A3 rapidly develop lethal acute myeloid leukemia (AML). We identified a long non-coding transcript in the NR4A3 locus and pursued the characterization of this anonymous transcript and the study of its role in leukemogenesis. We characterized this novel long non-coding transcript as a sense polyadenylated transcript. Bone marrow cells from AML patients expressed significantly reduced levels of lncNR4A3 compared to healthy controls (controls = 15, MDS= 20, p=0.05., AML= 21, pNR4A3, as previously reported, was also significantly reduced in AML. Interestingly, the expression of both coding and non-coding transcripts was highly correlated (Pearson R = 0.3771, PNR4A3, reduction of proliferation in myeloid cell lines K-562 and KG1 (n=3 and 2 respectively, pNR4A3 and we hypothesize this regulatory mechanism is mediated by the modulation of the RNA processing machinery.

Chronic myeloid leukemia (CML) is successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Insulin receptor substrate 1 (IRS1) has been shown to constitutively associate with BCR-ABL1, and IRS1-specific silencing leads to antineoplastic effects in CML cell lines. Here, we characterized the efficacy of NT157, a pharmacological inhibitor of IGF1R-IRS1/2, in CML cells and observed significantly reduced cell viability and proliferation, accompanied by induction of apoptosis. In human K562 cells and in murine Ba/F3 cells, engineered to express either wild-type BCR-ABL1 or the imatinib-resistant BCR-ABL1

INTRODUCTION Recent knowledge on the pathophysiology of sickle cell disease (SCD) have emphasized the role of hemolysis and nitric oxide (NO) depletion on the occurrence of acute and chronic complications. This new paradigm raises the possibility of innovative therapeutic approaches, including arginine supplementation. Areas covered: This review comments on the role of NO in the regulation of vascular tone, as well as its impaired metabolism in hemolytic diseases. Disturbances in these processes in SCD were detailed considering the functions of endothelial nitric oxide synthase (eNOS), arginase and asymmetric dimethylarginine (ADMA). Therapeutic approaches involving these pathways were discussed with emphasis on the effects of arginine therapy on the normalization of NO levels and its consequent clinical effects, mainly the decrease in the intensity of vaso-occlusive crises. Expert opinion: Considering the complex pathogenesis of the disease and the restricted access to curative therapies, the management of SCD must rely on a combination of therapies covering multiple pathways. Arginine supplementation, a low-cost approach, has shown promising results, which is particulary important considering most of the affected patients still live in unfavorable socioeconomic conditions. These findings should encourage further clinical trials, evaluating other outcomes and specific subpopulations, such as adult patients and compound heterozygotes.

RhoA and RhoC contribute to the regulation of glutamine metabolism, which is a crucial determinant of cell growth in some types of cancer. Here we investigated the participation of RhoA and RhoC in the response of prostate cancer cells to glutamine deprivation. We found that RhoA and RhoC activities were up- or downregulated by glutamine reduction in PC3 and LNCaP cell lines, which was concomitant to a reduction in cell number and proliferation. Stable overexpression of wild type RhoA or RhoC did not alter the sensitivity to glutamine deprivation. However, PC3 cells expressing dominant negative RhoA(N19) or RhoC(N19) mutants were more resistant to glutamine deprivation. Our results indicate that RhoA and RhoC activities could affect cancer treatments targeting the glutamine pathway.

LEF1 antisense RNA 1 (LEF1‐AS1) is an antisense long non‐coding RNA encoded in the lymphoid enhancer‐binding factor 1 (LEF1) locus. LEF1‐AS1 is a conserved transcript dysregulated in hematopoiesis. This study aimed to functionally characterize the role of this transcript in myeloid malignancy and explore a possible regulatory effect of LEF1‐AS1 upon LEF1. We show that LEF1‐AS1 is highly expressed in normal hematopoietic stem cells but barely detectable in myeloid malignant cell lines. Additionally, bone marrow cells from myelodysplastic syndrome (n=12) and acute myeloid malignancy patients (n=28) expressed significantly reduced levels of LEF1‐AS1 compared to healthy controls (n=15). Artificial LEF1‐AS1 over‐expression inhibited proliferation in HL60 and led to an upregulation of tumor suppressors p21 and p27, and reduced ERK1/2 activation. Unexpectedly, no underlying modulation of LEF1 was detected. Ectopic expression of LEF1‐AS1 also inhibited proliferation in HELA, a cell line lacking endogenous expression of LEF1, supporting a LEF1‐independent mechanism. Additionally, transient over‐expression of LEF1‐AS1 in AML patient cells also led to reduced proliferation and colony formation capacity. We used a mass spectrometry‐based proteomics approach. Proteomic quantification identified the modulation of an important metabolic regulator, Fumarase, and concomitant accumulation of the metabolite fumarate.

Disturbances in the physiological regulation of erythropoietin (EPO) in patients with sickle cell disease (SCD) may contribute to worsening anaemia and increased transfusion requirements, but the use of recombinant EPO in this group of patients is controversial. The objective of this study was to evaluate the use of this drug in adult patients with SCD and its effects on haemoglobin levels and transfusion requirements. We conducted a retrospective analysis at the University of Campinas, with nineteen adults with a diagnosis of SCD (HbSS and HbS/β+ thalassaemia), who had received at least 1 year of EPO therapy between 2007 and 2014. Haemoglobin concentrations and trends of variation in transfused RBC volumes were compared before and after EPO administration. We observed that seven patients had a good response to treatment (Hb increment higher than 1·5 g/dl) and nine had a partial response (0·5-1·5 g/dl increment) and there was a significant decrease in the need for transfusion amongst those who usually required regular transfusions. There were no increases in the rates of vaso-occlusive crisis or venous thromboembolism in comparison to the year before the onset of the therapy. Erythropoietin therapy led to a marked increase in haemoglobin concentration with a concomitant decrease in the demand for transfusion. Considering all complications related to allogeneic transfusion, we believe that EPO therapy represents an important therapeutic tool in sickle cell anaemia.

The endothelium plays an important role in sickle cell anemia (SCA) pathophysiology, interacting with red cells, leukocytes and platelets during the vaso-occlusive process and undergoing activation and dysfunction as a result of intravascular hemolysis and chronic inflammation. Blood outgrowth endothelial cells (BOECs) can be isolated from adult peripheral blood and have been used in diverse studies, since they have a high proliferative capacity and a stable phenotype during in vitro culture. This study aimed to establish BOEC cultures for use as an in vitro study model for endothelial function in sickle cell anemia. Once established, BOECs from steady-state SCA individuals (SCA BOECs) were characterized for their adhesive and inflammatory properties, in comparison to BOECs from healthy control individuals (CON BOECs). Cell adhesion assays demonstrated that control individual red cells adhered significantly more to SCA BOEC than to CON BOEC. Despite these increased adhesive properties, SCA BOECs did not demonstrate significant differences in their expression of major endothelial adhesion molecules, compared to CON BOECs. SCA BOECs were also found to be pro-inflammatory, producing a significantly higher quantity of the cytokine, IL-8, than CON BOECs. From the results obtained, we suggest that BOEC may be a good model for the in vitro study of SCA. Data indicate that endothelial cells of sickle cell anemia patients may have abnormal inflammatory and adhesive properties even outside of the chronic inflammatory and vaso-occlusive environment of patients.

Patients with severe leukocyte G6PD deficiency may present with impairment of NADPH oxidase activity and a history of recurrent infections, mimicking the phenotype of chronic granulomatous disease. We report herein a child with recurrent infections who initially received the diagnosis of G6PD deficiency. His erythrocyte G6PD activity was reduced: 1.8 U/g Hb (normal: 12.1 +/- 2.1 U/g Hb). Further studies revealed that G6PD activity in neutrophils, mononuclear leukocytes, and Epstein-Barr virus-transformed B-lymphocytes from the proband was similar to healthy controls. Molecular studies showed that the G6PD deficiency was due a 202 G-->A mutation, the A- variant common in African ethnic groups. The proband also exhibited severely impaired respiratory burst activity, as observed in X-linked CGD. Sequence analysis of genomic DNA showed a 264 G-->A substitution at the 3' splice junction of gp91-phox exon 3. The cDNA sequence showed a deletion of gp91-phox exon 3, giving rise to an unstable or nonfunctional mutant gp91-phox and to the phenotype of X-linked CGD. We propose that clinicians treating a patient with G6PD deficiency during a severe infection episode consider the possibility of temporary or permanent impairment of the phagocytes' microbicidal activity and the eventual association of G6PD deficiency and chronic granulomatous disease.

Evaluation of: Fibach E, Tan E, Jamuar S, Ng I, Amer J, Rachmilewitz EZ. Amelioration of oxidative stress in red blood cells from patients with β-thalassemia major and intermedia and E-β-thalassemia following administration of a fermented papaya preparation. Phytother. Res. DOI: 10.1002/ptr.3116 (2010) (Epub ahead of print).This article comments on the results obtained by Fibach et al., which showed reduction of oxidative status in red blood cells of patients with β- and E-β-thalassemia (β-thal) treated with fermented papaya preparation. The study was a three-center, prospective study, including eight patients with β-thal intermedia, four β-thal major and seven E-β-thal patients. The patients received 3 g of fermented papaya preparation (FPP) two- to three-times a day after meals, respectively, for 3 months. A marked decrease in reactive oxygen species, lipid peroxidation and phosphatidylserine externalization and an increase in GSH were detected in both groups of patients, indicating that FPP is efficien...

Background Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods In this study, gene expression profiles of CD34+ cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ≤ 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ≤ 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34+ cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

The aim of this work was to evaluate the regulation of SIRP alpha, an inhibitory phagocyte receptor, and the phosphatase SHP-1 in monocytes of patients with autoimmune hemolytic anemia, and the role of dexamethasone on SIRP alpha and SHP-1 gene expression and erythrophagocytosis in vitro. SIRP alpha and SHP-1 expression was higher in monocytes from AIHA patients compared with normal, returning to normal after glucocorticoid therapy. SIRP alpha and SHP-1 mRNA expression was upregulated in healthy monocytes treated with dexamethasone compared with basal; however, the erythrophagocytic ability was not altered. Our results point to a minor role of SIRP alpha and SHP-1 in determining AIHA.

Abstract 2133 The endothelium plays an important role in the pathophysiology of sickle cell anemia (SCA) due to interactions of endothelial cells with other cell types in the vaso-occlusive process and endothelial dysfunction caused by chronic inflammation and decreased nitric oxide bioavailability. Inflammatory molecules, such as cytokine TNF-α, may alter the endothelial cell surface and, thus, modulate the adhesion molecules that bind red blood cells (RBC). Damaged or activated endothelium produces cytokines and chemokines that result in the high levels of circulating inflammatory molecules found in the SCA. Blood Outgrowth Endothelial Cells (BOEC) are circulating endothelial cells isolated from the peripheral blood of adults, they have a high proliferative capacity in vitro, with the characteristic phenotype of mature cells. BOEC cultures were established to serve as a model for an in vitro study of endothelial properties and function in SCA, to evaluate RBC adhesion, expression of adhesion molecules and production of inflammatory cytokines in BOEC from healthy controls (Con) and SCA patients. Three cultures of BOEC from Con and three cultures from SCA patients in steady state were established and identification confirmed by flow cytometry (CD31+; CD146+; VEGF R2+; CD45-; CD133-). BOEC were used under basal conditions or following treatment with 10ng/ml TNF-α (3 H). RBC were isolated from Con, SCA and SCA patients on hydroxyurea treatment (SCAHU; 20–30 mg/Kg/day) and their adhesion to confluent Con and SCA BOEC was evaluated (4×106 cell/ml in EGM-2), using static adhesion assays. BOEC expression of adhesion molecules VCAM-1, E-selectin, P-selectin, CD36, the Vitronectin receptor and BCAM/Lu was evaluated by flow cytometry. The production and release of GM-CSF and IL-8 to the supernatant of BOEC in the presence and absence of an antiinflammatory stimulus (1μM Simvastatin, 3.5H) was quantified by ELISA. Notably, Con RBC adhered significantly more to SCA BOEC (16.6 ± 4.6%; n=14; p Support by FAPESP and CNPq Disclosures: No relevant conflicts of interest to declare.

Abstract 899 Sickle cell disease (SCD) results from mutations in the β chain of hemoglobin and is associated with a complex pathophysiological process that involves endothelial activation, inflammation and oxidative stress. Several clinical aspects of SCD such as pulmonary hypertension, moyamoya vasculopathy, proliferative retinopathy and leg ulcers suggest the involvement of an unbalanced angiogenesis. Plasma levels of vascular endothelial growth factor, placenta growth factor, angiopoietin-1 and angiopoietin-2 are reportedly elevated in SCD, although the effects of these alterations on angiogenic mechanisms in SCD are unclear. Hydroxyurea (HU) has been used successfully for SCD therapy and may act through several mechanisms. The purpose of this study was to evaluate the effects of plasma obtained from SCD patients upon capillary-like structure formation of human umbilical vein endothelial cell (HUVEC) cultures on Matrigel. Patients (in steady state) and controls were divided into six groups: HbSS phenotype not treated with HU (SSHU−), HbSS treated with HU (SSHU+), HbSC phenotype with retinopathy not treated with HU (SCR+HU−), HbSC without retinopathy and not treated with HU (SCR-HU−), HbSC with retinopathy and treated with HU (SCR+HU+) and control individuals (HbAA). Patients on HU had been taking 20–30mg/kg/day for at least 3 months. To assess tube formation, HUVECs (5× 104cells/mL) were cultured on growth factor reduced-Matrigel coated plates in the presence of 10% plasma and the number of cord-like structures quantified. Photomicrographs were taken using a phase-contrast inverted microscope. HUVEC were also incubated with HU (50–200 μM) and migration (double-chamber assay), proliferation (BrdU assay) and capillary-like structure formation (Matrigel) were addressed to compare effects of plasma from patients treated with HU to medium containing HU alone. After 17 H of incubation, a small decrease in HUVEC tube formation (4.1 ±4.9%, n=5, P>0.05 comp. to control, HUVEC in medium alone) was observed in the presence of HbAA group plasma, in agreement with reports suggesting an antiangiogenic activity of human plasma in absence of diseases. In contrast, angiogenesis of HUVEC increased significantly in the presence of plasma from all SCD patients, independently of retinopathy, as demonstrated by increases in tube formation indexes of 32.5 ±7.5%, 15.8 ±9.2%, 30.0 ±4.4% (N=5, P Disclosures: No relevant conflicts of interest to declare.

Abstract 3195 The hereditary persistence of fetal hemoglobin (HPFH) is the consequence of impaired switching in adult life, which results in the continued expression of gamma globin gene. The Brazilian HPFH type is characterized by a C → G substitution at the –195 position of the A gamma globin gene promoter, and associated with HbF levels ranging from 6 to 16% in the heterozygote state. This study was undertaken to identify genes that may be involved in hemoglobin switching and/or maintenance of elevated HbF levels in Brazilian HPHF subjects. The Suppressive Subtractive Hybridization libraries were constructed using a pool of RNA extracted from reticulocytes of peripheral blood in individuals with normal hematological data and from subjects with Brazilian and 55 and 57 overexpressed genes were identified in the normal and Brazilian HPFH library, respectively; findings were validated by qRT-PCR and Western blotting. One transcription factor identified was FOXO3a, whose expression was increased in Brazilian HPFH subjects, compared to control subjects. Moreover, the non-phosphorylated Foxo3a protein that interacts with DNA, was detected only in Brazilian HPFH when analyzed by Western blotting. FOXO3a binds to PAX1 promoter, a transcription factor whose activity was higher in Brazilian HPFH. The FOXO3a/PAX1 complex may be important in the mechanism responsible for increasing HbF levels in this genetic disorder. Another gene analyzed was KLF1, a transcription factor known as a regulator of switching from the gamma to beta globin gene. This gene was underexpressed in the reticulocytes of HPFH subjects. Klf1 protein activity in erythroid cells of healthy donors was also increased compared to Brazilian HPFH, when analyzed by DNA/protein array. Results suggest that the decreased KLF1 levels in Brazilian HPFH favors the interaction between the A gamma globin gene and the Locus Control Region, agreeing with previous studies that demonstrated that knockdown of KLF1 in adult erythroid progenitors favors the formation of complexes that bind to the gamma globin gene promoter. MIER1 expression was also found to be decreased in Brazilian HPFH reticulocytes, compared to controls. The MIER1 gene is able to recruit chromatin remodeling-enzymes leading, to the formation of heterochromatin and, consequently, silencing genes. This MIER1 may be an important gene in gamma to beta globin gene switching, where it could help in the maintenance of a closed chromatin structure in the gamma globin gene in individuals with low HbF levels. The expression of the HOOK3 gene was decreased in Brazilian HPFH reticulocytes, compared to controls. The HOOK3 encodes a protein that interacts with a GTPase protein, stimulating INF-gamma, responsible for the phosphorylation of p65/p50 and RXR beta like proteins which regulate the transcription of the beta globin gene. The reduced beta globin gene expression in Brazilian HPFH resulting from the reactivation of gamma globin gene may be responsible for a decrease in the HOOK3 expression. These results suggest that, in the HPFH Brazilian type, the FOXO3a/PAX1 complex could contribute to the continued expression of the A gamma globin gene. Additionally, some cellular modifications, such as low Klf1 activity and decreased HOOK3 and MIER1 expression, could participate in the maintenance of HbF levels. These genes could be important in therapeutic approaches for the development of new HbF induction agents for the hemoglobinopathies. Support by FAPESP, CNPq.and INCTS Disclosures: No relevant conflicts of interest to declare.

Abstract 2651 Background: LIGHT (TNFSF14; CD258), a recently-identified member of the TNF superfamily, is found associated with and produced from platelets, amongst other immune cells. Increased circulating levels of this protein have been observed in patients with myocardial infarction and acute atherothrombotic stroke and LIGHT has been proposed as a potential therapeutic target in atherosclerosis, due to its reported pro-thrombotic and pro-inflammatory effects upon human endothelial cells. This study evaluated whether production of LIGHT is altered in patients with sickle cell anemia (SCA), and the participation that the platelets (PLTs) may have in this production, since SCA is characterized by a significant chronic inflammation and endothelial activation that may initiate the vaso-occlusive process. Patients and Methods: Soluble LIGHT (sLIGHT) was determined in PLT-free plasma (obtained by sequential centrifugations and ultrafiltration) from healthy control individuals (CON), SCA patients in steady state (SCA) and SCA patients on hydroxyurea therapy (SCAHU; 20–30 mg/kg/day HU) by ELISA. Expressions of PLT-membrane LIGHT and PLT activation markers were evaluated by flow cytometry using anti-CD258-PE, anti-CD62P-FITC or anti-PAC1-FITC. Subjects had not taken ASA during the previous 14 days. Results: sLIGHT was significantly elevated in the plasma of SCA and SCAHU individuals, compared to CONs (SCA; 35.4 ± 9.4 pg/ml; SCAHU, 37.3 ± 7.5 pg/ml; CON, 9.7 ± 1.5 pg/ml, n=27, 27, 19, resp.; P0.05); basal and collagen-stimulated sLIGHT release correlated significantly with CD40L release (rp=0.654; rp=0.764, resp. P Conclusion: The pro-inflammatory and atherogenic protein, LIGHT, is found significantly elevated in the plasma of SCA patients. The correlation of plasma LIGHT with IL-8 indicates that LIGHT may participate in, or reflect, endothelial activation, whilst the correlation of circulating LIGHT and PLT release of LIGHT with CD40L indicates that the production of these two proteins may be tightly coupled. LIGHT protein is highly expressed on the PLT surface in SCA and this expression appears to be associated with PLT activation, rather than PLT number. Interestingly, HU therapy was not associated with any significant alteration in circulating LIGHT, nor PLT surface LIGHT. Future investigations will determine the extent of the contribution of LIGHT to endothelial activation, inflammation and vaso-occlusion in SCA and whether this protein holds promise as a potential therapeutic target for the disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 1030 FMNL1 belongs to a conserved family of formin-related proteins, has high restricted expression in malignant lymphoid derived cells and has been described as a tumor associated antigen in chronic lymphoid leukemia (CLL). Overexpression of FMNL1 in CLL has already been show to correlate with adverse clinical and biological parameters, particularly in young patients (E Calpe et al, ASH 2006, abstract submitted). Formins are known for their ability to act as effectors of Rho family GTPases, proteins that regulate multiple pathways involved in cell organization, adhesion, and proliferation. Deregulation of Rho GTPase family members has been associated with multiple human hematologic diseases such as leukemia. FMNL1 function and regulation have not yet been well characterized; its restricted expression suggests that FMNL1 represents an attractive target for novel immunotherapies in hematopoietic malignant disorders. Herein, we evaluated the association of FMNL1 with the three most studied Rho GTPase proteins (Rac1, RhoA and Cdc42) and the role of FMNL1, by gene silencing, in proliferation, migration and cell cycle in acute lymphoblastic leukemia cell lines. We also tested the cumulative effect of FMNL1 silencing and rapamycin treatment in proliferation assays. FMNL1 association with Rho GTPases proteins was analyzed by immunoprecipitation, followed by western blot. To inhibit FMNL1, specific shRNA-expressing lentiviral vector targeting the FMNL1 gene or no specific sequence (control) were used. Cell growth was measured using the MTT colorimetric reduction method in cells treated or not with different concentrations of rapamycin (10 or 100nM) in five replicates. Migration assays were performed in triplicate using 5-mm Transwells and the lower compartment was filled with 600 mL 0.5% BSA/RPMI containing 100ng/mL SDF-1. The experiments were performed in two cell lines: Jurkat and Namalwa. P value Disclosures: No relevant conflicts of interest to declare.

Abstract 2656 The pathophysiology of sickle cell anemia (SCA) is now recognized to include a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion that characterize this disease. The vaso-occlusive process is a multi-step process that involves different cell types, such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. In vivo transgenic animal studies indicated that the neutrophil appears to be the most important leukocyte in vaso-occlusion in previous transgenic animal in vivo studies; however, eosinophils (EOs) may also participate in this phenomenon in humans. EOs are found in significantly elevated numbers in sickle cell disease individuals and, in static adhesion assays, demonstrate augmented adhesive properties suggesting an activated state of these cells. However, the role of EOs in the pathophysiology of SCD and the effect of hydroxyurea (HU) therapy on the functional properties of these cells are not well understood. The aim of this study was to evaluate EO adhesion, migration and degranulation in SCA patients and the effect of HU therapy on these cells. EOs were purified from the peripheral blood of healthy controls (CON) (n≥10), steady-state SCA patients (n≥12) and SCA patients on HU therapy (n≥15), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion was determined by static adhesion assays and degranulation was evaluated by measurement of eosinophil peroxidase activity. Migration of EOs was determined in 96-multiwell chemotaxis chamber assays. Plasma levels of Eotaxin-1, 2 and RANTES, important eosinophil attracting chemokines, were determined by ELISA. The absolute number of EOs in the peripheral blood of SCD patients not treated with HU was significantly higher compared to control individuals (0.504±0.09 vs 0.188±0.04; P=0.01, respectively). SCA patients taking HU presented significantly lower numbers of EOs (0.200±0.05, P = 0.01). Basal adhesion of EOs from SCA patients was significantly higher than in healthy controls (22.8±2.6 vs 13.7±1.7%, respectively, P = 0.004). Furthermore, the EOs from SCA patients on HU therapy demonstrated a significantly lower adhesion to fibronectin (12.5±2.0%, P=0.002). Spontaneous EO chemotaxis was significantly increased in SCA patients, compared to healthy controls (12.9±1.56 vs 6.97±0.8 × 105/ml, respectively, P=0.009). Interestingly, the chemotatic response of EOs isolated from SCA patients taking HU was also higher than that of control EOs (12.1± 1.9 × 105/ml). EO chemotaxis in response to RANTES, Eotaxin and IL5 was significantly augmented in all groups compared to spontaneous chemotaxis (RANTES: CON: 15.3± 2.3; SCA: 19.9±3.0; SCAHU: 25.0±4.7; Eotaxin: CON: 18.7±3.0; SCA: 24.5±4.2; SCAHU: 24.0±5.2; IL5: CON: 13.4±2.4; SCA: 18.8±3.0, SCAHU: 16.6±4.5, P Disclosures: No relevant conflicts of interest to declare.

Abstract 1886 Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia (AML). The abnormal differentiation of myeloid and erythroid cells is probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling, from upstream activators, to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R) and Erythropoietin receptor (EPOR). It is upregulated and phosphorylated by EPO in normal bone marrow erythroblasts and in UT-7 leukemic cells, as well as in HL60 leukemic cells during granulocytic-differentiation upon DMSO induction. IGF1 signaling is capable of inducing differentiation in several cell types and plays an important role in the regulation of human erythropoiesis. EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. In view of the role of IRS2 in the erythroid and granulocytic differentiation process, we hypothesized that IRS2 might be related to the deficient differentiation of MDS cells and disease progression. The aim of the present study was to characterize the mRNA and protein expression levels of IRS2 in cells of MDS patients and normal donors and to analyze the IRS2 expression levels between low-risk and high-risk of MDS. We also elucidated the expression levels of IRS2 transcripts during erythroid differentiation of CD34+ cells from normal donors and MDS patients. We studied 12 healthy donors and 29 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification; 15 low-risk [RCUD/RCMD/RARS] and 11 high-risk [RAEB-1/RAEB-2] according to WHO classification; 22 low/INT-1 risk and 7 INT-2/high risk according to IPSS; 25 low risk and 4 intermediate/high risk cytogenetic). RT-PCR was performed in total cell from bone marrow samples for gene expression studies. Protein expression was evaluated by Western blot in bone marrow mononuclear cells from MDS patients or in peripheral blood CD34+ from normal donors. Erythroid-differentiation was performed in CD34+ bone marrow cells from 4 normal donors and 4 MDS patients. IRS2 gene expression was significantly decreased in primary MDS cells compared with normal cells (0.74 [4.06-0.15] vs. 4.71 [11.78-0.66], P Disclosures: No relevant conflicts of interest to declare.

Abstract 2943 Background. Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by ineffective and dysplastic hematopoiesis, progressive bone marrow failure, cytopenias and a high risk of transformation into acute leukemia. Thrombocytopenia is detected in up to two thirds of patients with MDS and severe thrombocytopenia is present in approximately 10%. Besides ineffective thrombopoiesis, immune destruction of platelets could be an additional factor in the genesis of thrombocytopenia, since immunological abnormalities are also frequent in patients with MDS. The detection of platelet associated IgG (PAIgG) by immunofluorescence (platelet immunofluorescence test or PIFT) is a highly sensitive assay. In addition, some morphological platelet indices (PDW and MPV) are correlated with the occurrence of immune thrombocytopenia. We prospectively analysed platelet-bound IgG and platelet indices (PDW and MPV) in 35 patients with MDS. Methods: Thirty-five patients with MDS (mean age ± SD: 63 ± 19 yo; range 21–89 yo; 15female/20male) were evaluated. According to FAB, 27 patients were classified as RA, 5 as RARS and 3 as RAEB. Clinical manifestations of immunological disorder were not present in this population. Blood samples were analyzed by PIFT, in order to detect platelet associated IgG and results were expressed as a ratio of patient fluorescence/negative control fluorescence (R). Cell-dyn Sapphire blood cell analyzer (Abbott, Illinois, USA) was used to measure platelet count, mean platelet volume (MPV) and platelet size deviation width (PDW). Thrombocytopenia was defined as a platelet count Results. Platelet counts of the entire population ranged from 6.7 to 708 ×109/L, with median of 95.4 × 109/L. Eighteen patients (51.43%) had platelet count Conclusion: Thus, we propose that a combination of a simple index as MPV and a highly sensitive and easy to perform screening test for PAIgG as PIFT could be applied to select a subset of MDS patients in which we would be able to prevent the overuse of unnecessary platelet transfusions and who could be candidates for an immunosuppressive therapeutic approach. Supported by INCTS, FAPESP, CNPq. Disclosures: No relevant conflicts of interest to declare.

Abstract 4459 Background: Signaling pathway of the insulin-like growth factor 1/Insulin Receptor Substrates (IGF-I/IRS) plays an important role in the development of various cancers such as breast, colon and prostate cancer. IRS proteins, including IRS1, act on Akt/mTOR and MAPK pathways, through their interaction with effectors such as PI3K, SHP2 and Grb2. PI3K and Grb2 activation, leading to higher Akt and ERK1/2 phosphorylation, respectively, culminating in an increase of proliferation. It has already been described that IRS1 is overexpressed and negatively correlated with survival in ALL Philadelphia-positive; and IRS1 is constitutively phosphorylated and binds with BCR-ABL in K562 cells. However, the function of IRS1 in BCR-ABL positive cells remains an interesting issue to be clarified. The aim of the present study was to evaluate the effects of IRS1 knockdown upon K562, regarding proliferation and apoptosis, as well as the effects upon Akt/mTOR, MAPK, and BCR-ABL signaling pathways. Methods: Specific shRNA-expressing lentiviral vector targeting the IRS1 gene or no specific sequence (control) were used in the K562 cell line. Real-time RT-PCR and Western blot analysis were performed to determine the inhibition of IRS1 expression. After 48 hours of culture, proliferation was analyzed by MTT assays, apoptosis by Anexin-V and propidium iodide (PI), cell cycle by incubation with PI and RNase A buffer and flow cytometry. Western blot and immunoblotting with specific antibodies was used to evaluate Akt, P70S6K, ERK 1/2 and CRKL expression and phosphorylation. Immunoprecipitation and immunoblotting were used for evaluating BCR-ABL phosphorylation Results: The levels of IRS1 mRNA and IRS1 protein in IRS1 knockdown cells were significantly reduced by 83.7±2.26% and 73.6±9.07%, respectively (P Conclusion: IRS1 plays an important role in the proliferation of K562 cells and the lack of IRS1 decreased proliferation and Akt, P70S6K and ERK phosphorylation, indicating a downregulation of Akt/mTOR and MAPK pathways. The lack of synergism between imatinib treatment and IRS1 knockdown on proliferation and the lack of differences in BCR-ABL and CrkL phosphorylation in IRS1 knockdown suggest that IRS1 is downstream to the BCR-ABL pathway and plays an important role in Akt/mTOR and MAPK activation in Philadelphia-positive cells. This work was supported by FAPESP and CNPq. Disclosures: No relevant conflicts of interest to declare.

Abstract 2653 The antimicrobial peptide hormone hepcidin is a key regulator of iron metabolism. Although mainly produced in the liver, hepcidin is also known to be synthesized by monocytic-macrophagic cells. We have previously shown that hepcidin is overexpressed in mononuclear cells in patients with sickle cell anemia (SCA), but whether monocyte-derived hepcidin production is related to iron metabolism remains unknown. To gather further insight into the role of hepcidin in monocytes, we collected peripheral blood samples from adult SCA patients (n=54) and normal age- and race-matched controls (CTRL, n=13) for assessment of hematological parameters, biochemical markers of iron status (serum iron and transferrin saturation - TfSat), hemolysis (lactate dehydrogenase-LDH) and inflammation (C reactive protein - CRP), and separation of peripheral blood monocytes by Ficoll-Hypaque and Percoll gradients for gene expression analyses of genes involved in iron metabolism signaling (HAMP encoding hepcidin, and three genes belonging to different pathways known to influence hepcidin expression, STAT3, SMAD4 and TLR4). Plasma GDF-15 levels were also measured, as this protein has been shown to be a potent downregulator of hepcidin. SCA patients were further stratified according to the number of previous blood transfusions and to treatment with hydroxyurea (HU). All patients were in steady-state, had no history of iron chelation treatment and were not enrolled in a regular transfusion program. 18 patients were receiving HU and 15 from the non-HU group had received over 20 blood transfusions during their lifetime. As expected, SCA patients had elevated LDH levels, but no differences were found between control and SCA groups regarding serum iron, TfSat or CRP levels. Except for a higher red cell mean corpuscular volume, patients on HU did not differ significantly from patients not using HU. Plasma GDF-15 levels were higher in SCA patients (2146±506.4pg/mL) than in control individuals (228.5±21.0pg/mL, p90% viable cells at all experiments), and HAMP gene expression was increased up to four times in the cell cultures exposed to HU (p=0.03), while STAT3 and SMAD4 expressions remained unchanged. We have shown that hepcidin expression is upregulated in monocytes in SCA patients, particularly in those receiving HU, and that HU is capable of inducing this expression in an in vitro model, independently from inflammatory cytokine-mediated stimulation. Our data suggest that, although liver-derived hepcidin has been shown to have a major role in iron metabolism, its monocyte-derived counterpart does not seem to be directly influenced by iron status and may have other functions. Some studies have demonstrated that hepcidin in other species has anti-inflammatory effects in vitro, and that patients with SCA on HU shift to a lower inflammatory status. Thus, monocytic hepcidin overexpression might be a response against the chronic inflammatory state in SCA, and HU treatment may enhance this response. This is the first description of monocyte-derived hepcidin in SCA and the influence of HU on its expression. Disclosures: No relevant conflicts of interest to declare.