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Iron availability for erythropoiesis and its dysregulation in β-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in β-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin's effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in β-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that β-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective β-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in β-thalassemic mice. To evaluate further, we crossed TfR1+/- mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with β-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to β-thalassemic mice. Our data demonstrate for the first time that TfR1+/- haploinsufficiency reverses iron overload specifically in β-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during β-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in β-thalassemic mice.

Human babesiosis is a global emerging infectious disease caused by intraerythrocytic parasites of the genus Babesia. Its biology has remained largely unexplored due to a lack of critical tools and techniques required to define the various stages and phases of the parasite's cycle in its host RBC and the interplay between host and parasite. This article presents a powerful set of tools combining stage synchronization of the parasite with a platform that encompasses both a flow cytometric evaluation of the subpopulation structure of the parasite population together with a morphological assessment of the population parasites using light microscopy of conventional Giemsa stained smears. Together, these yield specific information on the effect of any drug/condition of interest and its targeted biological process, allowing the characterization of the adaptive response of parasites to a particular stressor agent. Three inhibitors were used in this study, each targeting a specific phase of the parasite's lifecycle, neuraminidase for host cell invasion, N-acetyl-L-leucyl-L-leucyl-L-norleucinal for parasite development and EGTA for parasite egress from the host cell. Results presented prove the power of this combination platform in discriminating the specific targets among the life-cycle processes of the parasite-invasion, development/proliferation and egress. This will expand the range of queries that can now be successfully addressed in this parasite, opening avenues for the development of new methods to control babesiosis, either by chemicals (screening for new chemotherapy drugs or defining levels of parasite resistance) or physical methods (light irradiation or heat shock used in pathogen reduction/elimination methods). © 2017 International Society for Advancement of Cytometry.

Babesia parasites cause a malaria-like febrile illness by infection of red blood cells (RBCs). Despite the growing importance of this tick-borne infection, its basic biology has been neglected. Using novel synchronization tools, the sequence of intra-erythrocytic events was followed from invasion through development and differentiation to egress. The dynamics of the parasite population were studied in culture, revealing for the first time, the complete array of morphological forms in a precursor-product relationship. Important chronological constants including Babesia's highly unusual variable intra-erythrocytic life cycle, the life span of each population of infected cells and the time required for the genesis of the different parasite stages were elucidated. Importantly, the maintenance of specific ratios of the infected RBC populations was shown to be responsible for the parasites' choice of developmental pathways, enabling swift responses to changing environmental conditions like availability of RBCs and nutrition. These results could impact the control of parasite proliferation and therefore disease.

T follicular helper cells are the main CD4(+) T cells specialized in supporting B-cell responses, but their role in driving transfusion-associated alloimmunization is not fully characterized. Reports of T follicular helper subsets displaying various markers and functional activities underscore the need for better characterization/identification of markers with defined functions. Here we show that a previously unidentified subset of human circulating T follicular helper cells expressing TIGIT, the T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains, exhibit strong B-cell help functions. Compared to the subset lacking the receptor, T follicular helper cells expressing this receptor up-regulated co-stimulatory molecules and produced higher levels of interleukins (IL-21 and IL-4) critical for promoting B-cell activation/differentiation. Furthermore, this subset was more efficient at inducing the differentiation of B cells into plasmablasts and promoting immunoglobulin G production. Blocking antibodies abrogated the B-cell help properties of receptor-expressing T follicular helper cells, consistent with the key role of this molecule in T follicular helper-associated responses. Importantly, in chronically transfused patients with sickle cell anemia, we identified functional differences of this subset between alloimmunized and non-alloimmunized patients. Altogether, these studies suggest that expression of the T-cell immunoreceptor with Ig and immunoreceptor tyro-sine-based inhibitory domains not only represents a novel circulating T follicular helper biomarker, but is also functional and promotes strong B-cell help and ensuing immunoglobulin G production. These findings open the way to defining new diagnostic and therapeutic strategies in modulating humoral responses in alloimmunization, and possibly vaccination, autoimmunity and immune deficiencies.

HIV protease inhibitors (HIV-PIs) are a class of antiretroviral drugs designed to target the viral protease. Strikingly, these drugs have also been reported to possess antitumor effect. In this study, we evaluated the activity of one HIV-PI, Nelfinavir, against human cervical cancer cells. We found that Nelfinavir inhibited the growth of cervical cancer cell lines at the lowest micromolar concentrations clinically attainable. Nelfinavir promoted apoptosis and arrested the cell cycle at G1 phase. Apoptosis is attributed to the promotion of mitochondrial reactive oxygen species (ROS) production, which results in the translocation of mitochondrial apoptosis inducing factor (AIF) to the nucleus. We further showed that Nelfinavir increased mitochondrial ROS production by decreasing manganese superoxide dismutase (MnSOD) protein levels. Taken together, our results suggest that Nelfinavir can be repositioned as a cervical cancer therapeutic.

Human babesiosis is a global emerging infectious disease caused by intraerythrocytic parasites of the genus Babesia. Its biology has remained largely unexplored due to a lack of critical tools and techniques required to define the various stages and phases of the parasite's cycle in its host RBC and the interplay between host and parasite. This article presents a powerful set of tools combining stage synchronization of the parasite with a platform that encompasses both a flow cytometric evaluation of the subpopulation structure of the parasite population together with a morphological assessment of the population parasites using light microscopy of conventional Giemsa stained smears. Together, these yield specific information on the effect of any drug/condition of interest and its targeted biological process, allowing the characterization of the adaptive response of parasites to a particular stressor agent. Three inhibitors were used in this study, each targeting a specific phase of the parasite's lifecycle, neuraminidase for host cell invasion, N-acetyl-L-leucyl-L-leucyl-L-norleucinal for parasite development and EGTA for parasite egress from the host cell. Results presented prove the power of this combination platform in discriminating the specific targets among the life-cycle processes of the parasite-invasion, development/proliferation and egress. This will expand the range of queries that can now be successfully addressed in this parasite, opening avenues for the development of new methods to control babesiosis, either by chemicals (screening for new chemotherapy drugs or defining levels of parasite resistance) or physical methods (light irradiation or heat shock used in pathogen reduction/elimination methods). © 2017 International Society for Advancement of Cytometry.

Background: Increased baseline white blood cell count (WBC) has been associated with vaso-occlusive complications in sickle cell disease (SCD), raising concern for the use of mobilizing agents that increase WBC count for hematopoietic progenitor cell (HPC) collection for gene therapy. Indeed, G-CSF, which predominantly increases neutrophil counts, when administered to SCD patients can precipitate life-threatening vaso-occlusion. Such vaso-occlusion, however, does not always correlate with degree of white blood cell count elevation (Fitzhugh C et al, Cytotherapy 2009) and G-CSF is associated with increases in neutrophil activation and adhesion. This raises the question whether WBC might be a surrogate for other proposed contributors to vaso-occlusion, such as activation of white cells, platelets, and coagulation. We examined this issue in the context of a clinical study of HPC mobilization with a single dose of plerixafor alone in clinically stable SCD patients. Materials and Methods: 14 patients (13 SS, 1 SB0, median age 31 with range 21-46) have completed the study so far (6 patients at 80 ug/kg, 3 patients at 160 ug/kg, and 5 patients at 240 ug/kg). 10 patients were on hydroxyurea (HU) at a median dose of 25 mg/kg (range 16-28 mg/kg) with a mean HbF of 17% versus 7% for the non-HU treated patients. Only one patient (non-HU treated) had received recent red cell transfusion (for leg ulcers) and had HbA (54%) present. Whole blood was collected at means of 4 hr pre-dose and 12 hr post-dose for activation marker studies by flow cytometry (cell studies) or ELISA (coagulation studies) as listed in Table 1. Results: Increases in absolute total white cell, neutrophil, monocyte, and lymphocyte concentrations in whole blood with plerixafor treatment were all highly significant (Table 2, p < 0.002), without significant differences between HU- and non-HU treated patients or between dose levels. Twelve of 14 patients, however, did not experience any significant adverse events from plerixafor administration. Absolute neutrophil counts (ANC) were strongly correlated with absolute concentrations (but not percentages) of activated β2 integrin neutrophils (r= 1.0) and activated Mac-1 neutrophils (r= 0.8) but no other activation markers. We observed significant plerixafor-associated increases in activation markers (Figure 1) only for ANC subsets positive for activated β2 integrin (1.7± 0.44 fold, mean ± SD, p=0.002) and activated Mac-1 (1.7 ± 1.2 fold, p=0.05), which the correlations with ANC partly explain. The activated Mac-1 significance, however, disappeared with Bonferroni correction. There were no significant differences in activation markers between dose levels or between HU- and non-HU treated patients. There were moderate correlations between activated β2 integrin or activated Mac-1 and absolute reticulocyte count (r= 0.6 for both) and HbF (r= -0.5 for both) but not between these activation markers and LDH. Two patients had pain crises starting at 48 hr (80 ug/kg dose) and 81 hr (240 ug/kg dose) post-plerixafor administration; their pre and post-dose (12 hr) white counts are shown in Table 3. The fold-increase in activation markers for these 2 patients is summarized in Figure 2 (for each activation marker, color-coded arrows point to the two patients) Conclusion: As with G-CSF, elevation of WBC or their subsets with plerixafor is not necessarily associated with vaso-occlusion, as manifested by pain crises. In our study, however, ANC elevation correlated with absolute concentrations of neutrophils positive for activated β2 integrin and activated Mac-1, which were the only activation markers significantly increased with plerixafor. These activation markers in turn correlated with reticulocyte count and HbF, which influence SCD vaso-occlusive severity. Vaso-occlusion as manifested by pain crises, however, was not consistently associated with the highest fold increases in ANC or activation markers. Therefore it remains unclear which bioassays correlate with vaso-occlusion associated with plerixafor mobilization. Disclosures No relevant conflicts of interest to declare.

Babesia parasites cause a malaria-like febrile illness by infection of red blood cells (RBCs). Despite the growing importance of this tick-borne infection, its basic biology has been neglected. Using novel synchronization tools, the sequence of intra-erythrocytic events was followed from invasion through development and differentiation to egress. The dynamics of the parasite population were studied in culture, revealing for the first time, the complete array of morphological forms in a precursor-product relationship. Important chronological constants including Babesia's highly unusual variable intra-erythrocytic life cycle, the life span of each population of infected cells and the time required for the genesis of the different parasite stages were elucidated. Importantly, the maintenance of specific ratios of the infected RBC populations was shown to be responsible for the parasites' choice of developmental pathways, enabling swift responses to changing environmental conditions like availability of RBCs and nutrition. These results could impact the control of parasite proliferation and therefore disease.

Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for erythropoiesis, and TfR1 is found in highest concentrations on erythroid precursors due to high iron requirement for hemoglobin (Hb) synthesis. Diseases of ineffective erythropoiesis such as β-thalassemia, are characterized by anemia, expanded and extramedullary erythropoiesis, and iron overload. Iron overload results from insufficient hepcidin, a peptide hormone secreted by hepatocytes in response to iron load. In β-thalassemia, hepcidin is relatively suppressed as a consequence of erythroid expansion. Erythroferrone (ERFE), a recently described erythroid-derived hepcidin suppressor, has been proposed as the mechanism and found in higher concentration in bone marrow of β-thalassemic mice. We previous demonstrate that exogenous transferrin (Tf) ameliorates anemia in β-thalassemic mice, reversing splenomegaly, hepcidin suppression, and iron overload and recently confirmed a decrease in Erfe expression in erythroid precursors from Tf-treated β-thalassemic mice. We observed that although Tf-treated β-thalassemic mice exhibit a further decrease in MCV and MCH, suggesting a relatively more iron restricted erythropoiesis, TfR1 expression is decreased. We hypothesize that TfR1 is central to Tf's effect on erythropoiesis in β-thalassemic mice. Last year, we presented our analysis of th3/+ TfR1+/- double heterozygote mice which exhibit reversal of all erythropoiesis- and iron-related pathology in th3/+ mice, confirming our observations in Tf-treated β-thalassemic mice and further supporting our hypothesis. To evaluate the mechanism involved, we observed that despite suppressed TfR1 concentration in reticulocyte (P=0.006) and sorted bone marrow erythroid precursors (P=0.0004) from Tf-treated th1/th1 mice, cell surface TfR1 expression decreased on reticulocytes (P=0.003) but was surprisingly increased on late stage erythroid precursors (P=0.007) (Figure 1A), suggesting that exogenous Tf influences erythroid precursor enucleation. Because we previously demonstrate decreased serum soluble TfR1 in Tf-treated th1/th1 mice [Liu J Blood 2013], we hypothesize that exogenous Tf alters TfR1 shedding from erythroid precursor membranes, promoting enucleation and improved terminal differentiation. We observed decreased enucleation using syto60 in flow cytometry of fetal liver cells (FLC) from th3/+ relative to wild type (WT) embryos (35 vs. 51%, P=0.03) which is normalized by exposure of th3/+ FLCs to Tf in vitro (58 vs. 41%, P=0.001) (Figure 1B). Tf-treated th3/+ FLCs shed more TfR1 to the nuclear fraction relative to reticulocyte during enucleation (P=0.0001) (Figure 1C). Furthermore, enucleation isdecreased in vivo in th3/+ relative to WT FLCs and peripheral blood at E14.5 and normalized in th3/+ TfR1+/- double heterozygote mice (45 vs. 35%, P=0.002) (Figure 1D). Interestingly, we analyzed iron status in TfR1+/- mice revealing that serum hepcidin is increased relative to WT (323 vs. 190 ng/ml, P=0.04) despite minimally decreased serum and liver iron concentrations (no statistically significant differences) and increased Erfe expression in erythroid precursors (5-fold, P=0.04). Relative to th3/+ mice, double heterozygote mice exhibit decreased serum iron (94 vs. 133 ug/dl), non-heme liver iron (0.31 vs. 0.74 mg iron/g dry weight, P=0.02), and Erfe expression (0.3-fold, P=0.04). Although no difference is observed between double heterozygote mice and th3/+, serum hepcidin is significantly increased in double heterozygote mice compare to WT (392 vs. 190 ng/ml, P=0.01), suggesting a more appropriate hepcidin response to iron overload (Figure 1E). Taken together, we postulate that decreased TfR1 expression plays a critical role in reversing ineffective erythropoiesis by increasing enucleation and influences hepcidin regulation in an ERFE independent manner. Disclosures No relevant conflicts of interest to declare.

Low/intermediate risk myelodysplastic syndromes (MDS) is associated with relatively longer survival and high transfusion requirements, resulting in secondary iron overload. Iron overload is an independent variable associated with poor prognosis, impacting survival, and iron chelation therapy is associated with prolonged survival in transfusion-dependent MDS patients. We have previously shown that exogenous transferrin (Tf) results in more circulating red blood cells (RBCs), increased hemoglobin (Hb), reversal of splenomegaly, and improvement in ineffective erythropoiesis in β-thalassemic mice (Li Nat Med 2010) and recently demonstrate that erythroferrone (ERFE), a newly described physiologic erythroid regulator of hepcidin, is normalized in Tf-treated β-thalassemic mice (Chen, manuscript in review). We postulate that ineffective erythropoiesis in β-thalassemia shares many characteristics with that of low risk MDS, such as defective erythroid differentiation, anemia, splenomegaly, and systemic iron overload, suggesting that similar effects of exogenous Tf may be relevant also in MDS. Partial deletion of chromosome 5, 5q-syndrome, is the most common cytogenetic abnormality in low/intermediate risk MDS. We characterize a mouse model of 5q-syndrome, Cd74+/lox Nid67+/lox, Lmo2Cre+ mice (5q-mice) (Barlow Nat Med 2010). Our findings reveal that 5q-mice exhibit macrocytic anemia (Hb 6 vs. 12 g/dL, P Disclosures No relevant conflicts of interest to declare.

Background Immune Thrombocytopenia (ITP) is a bleeding disorder due to a combination of increased platelet destruction and reduced production, often secondary to anti-platelet/megakaryocyte antibodies. The presence of antibodies to glycoproteins (GP) IIb/IIIa (integrin αIIbβ3) and GPIb/IX, detected in majority of ITP patients, may correspond to different responses to treatment, i.e., anti-GPIb is associated with more severe disease, and less responsive to intravenous immunoglobulins and steroids. Thrombopoietin Receptor Agonists (TPO-RA) increase platelet production by stimulation of megakaryopoesis. Predictors of response to TPO-RA and influence of antibody profile on response are currently unknown. In our previous study we investigated Absolute Immature Platelet Fraction (A-IPF) prior to TPO-RA treatment and did not find a correlation between A-IPF, anti-GP antibodies, and platelet counts. The aims of this study were to further investigate: 1. The role of anti-GP antibodies in response to TPO-RA; 2. Effect of patients' antibodies on megakaryocyte (MK) viability, maturation, apoptosis and formation of proplatelets (in vitro); 3. The influence of patients' clinical characteristics on response to TPO-RA. Materials and Methods 91 patients with persistent or chronic ITP, were treated at Weill Medical College of Cornell University until January 2015 with TPO-RAs: 52 patients received eltrombopag, 22 romiplostim and 17 avatrombopag. Serum samples of 84 patients were analyzed for the presence of anti-GP by MAIPA assay as previously described. Patients with baseline platelet counts less than MKs were derived from human umbilical cord blood stem cells as previously described. Cells were grown using SFEM medium, adding on day 0 of culture 50 ng/ml recombinant TPO and aliquots of serum of ITP patients or healthy controls. The percentages of immature (CD41+/CD42-), mature (CD41+/CD42+), viable and apoptotic MKs were analyzed by flow cytometry on day 12. Apoptosis was analyzed by measuring Mitochondrial Outer Membane Potential (MOMP) and Phosphatidyl Serine (PS) externalization. MKs were considered apoptotic if they had positive staining for PS externalization, viable if positive for MOMP, and dead if positive for 7-Aminoactinomycin D (7AAD). Proplatelet formation by MKs was analyzed by microscopy. Statistical analysis using unpaired T-test and Pearson correlation test were performed. Results Ninety-one patients were included, 40 male (44%) and 51 female (56%), with a median age of 37.4 years (range 2-87). Median duration of ITP before TPO-RA treatment was 8 years (range 0.3-45). The 18/91 (19.8%) non-responders to TPO-RA were not different from the 73/91 responders in age, gender, number of prior treatments, duration of ITP, and past splenectomy. The presence of either or both anti-GP antibodies was correlated with average lower platelet counts on TPO-RA: 82.3 x109/L versus 123x109/L in patients without detected antibodies ("neither") (p=0.003). However, the response to TPO-RA was not influenced by the type of antibody: in patients with anti-GPIb the average platelet count was 76.1x109/L, and with anti-GPIIb/IIIa 80.7x109/L (Figure 1). In culture, excess dead MKs were found in anti-GPIb group and antiGPIb&antiGPIIb/IIIa group compared to "neither" group (p=0.0013 and p=0.027 respectively) and comparing antiGPIb&antiGPIIb/IIIa to control (p=0.0025). We did not observe changes in the degree of MK apoptisis or in MK maturation in the presence of serum antibodies. In cultures treated with serum of patients having anti-GPIb, less proplatelets were detected comparing to control (p=0.044) or to "neither" (p=0.0039). We conclude that patients with anti-GP antibodies respond less to TPO-RA, however there is no difference in response to TPO-RA between patients having anti-GPIb and anti-GPIIb/IIIa, unlike responses to other treatment modalities (e.g., steroids or immunoglobulins). TPO-RA could be a preferable treatment option in ITP patients having anti-GPIb. Figure 1. Average 6-months platelet counts of TPO-RA-treated ITP patients divided into groups by presence of antibody/ies. Figure 1. Average 6-months platelet counts of TPO-RA-treated ITP patients divided into groups by presence of antibody/ies. Disclosures Off Label Use: Eltrombopag, romiplostim and avatrombopag are a thrombopoietin receptor agonist approved for the treatment of thrombocytopenia in adults with chronic ITP. In some preliminary studies these medicines found as safe and effective treatment option in children and adolescents. Bussel:amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Novartis: Consultancy, Research Funding; Genzyme: Consultancy; BiologicTx: Research Funding; Ligand: Consultancy, Research Funding; Eisai: Consultancy, Research Funding; Shionogi: Consultancy, Research Funding; momenta: Consultancy; Protalex: Consultancy; Symphogen: Consultancy.

β-thalassemia is an inherited blood disorder caused by reduced or absence of β-globin expression which results in imbalanced globin synthesis, ineffective erythropoiesis, and anemia. How the imbalance between α- and β-globin results in ineffective erythropoiesis, if apoptosis or dysfunctional differentiation of erythroid precursors results in ineffective erythropoiesis, and whether disrupted iron regulation and / or iron overload in β-thalassemia is directly involved in the pathophysiology of ineffective erythropoiesis is incompletely understood. Iron is critical for hemoglobin synthesis and erythropoiesis is dependent on transferrin (Tf) bound iron. Tf functions as the main iron transporter in circulation, where it exists in three forms: as iron-free apo-transferrin (apoTf), monoferric Tf, or diferric Tf (holoTf). Typically, iron is bound to 30% of all Tf binding sites in circulation at which point monoferric Tf is found in the highest concentration relative to holoTf. . We have previously shown that exogenous apoTf ameliorates anemia in a mouse model of β-thalassemia intermedia (th1/th1), resulting in reduced splenomegaly, reticulocytosis, and α-globin precipitation on circulatory red blood cells (RBC). We also observe a decrease in mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), serum iron, Tf saturation, together suggesting that relative iron deficiency improves iron metabolism and ineffective erythropoiesis in apoTf-treated th1/th1 mice. We hypothesize that exogenous apoTf decreases cytosolic iron and heme as a consequence of increased monoferric Tf which results in less iron entering cells via Tf:TfR1 binding. Our current data reveals that in vitro incubation of purified apoTf and holoTf at 37C results in the formation of monoferric Tf, and injection of wild-type (WT) mice with a single intraperitoneal dose of apoTf (10mg) decreases holoTf (P=0.01) and increases monoferric Tf (P=0.02) in the serum 6 hours after injection. Using both calcium mobilization and anti-Tf antibodies in flow cytometry, we demonstrate that apoTf results in no TfR1 binding relative to holoTf in CHO cells (P Disclosures No relevant conflicts of interest to declare.

Iron overload causes morbidity and mortality in patients with β-thalassemia. Transfusion independent patients develop iron overload from increased dietary iron absorption, implicating inappropriately low hepcidin and supporting the therapeutic potential of approaches to increase hepcidin. Relatively low liver hepcidin mRNA expression is also characteristic of mouse models of β-thalassemia, and a recently identified “erythroid factor,” erythroferrone, has been implicated in hepcidin suppression in thalassemia. We have previously shown that exogenous apo-transferrin injections result in relatively iron restricted erythropoiesis, ameliorate ineffective erythropoiesis, and increase hepcidin expression in Hbbth1/th1 β-thalassemia intermedia (thalassemic) mice. We now explore the effect of exogenous apo-transferrin on signaling pathways (i.e. Smad and Erk) and circulating parameters thought to participate in hepcidin regulation in vivo and in vitro in wild type (WT) and thalassemic mice. Our results demonstrate that apo-transferrin injection increase both serum hepcidin concentration (603 vs. 306 ng/ml, n=13-24 per group, P Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Westerman: Intrinsic Lifesciences LTD: Employment, Equity Ownership.

Transferrin receptor 1 (TfR1) is found in highest concentrations on erythroid precursors due to the disproportionately high iron requirement for hemoglobin synthesis, making transferrin-bound iron binding to TfR1 essential for erythropoiesis. Recent data reveals that TfR1 mRNA expression (6.48±2.23 vs. 1.0±0.25 relative to GAPDH, P=0.04 in sorted basophilic erythroblasts), whole cell protein concentration measured using ImageJ (11496±1783 vs. 1620±1448, P=0.0001 in reticulocytes), and cell surface concentration measured using flow cytometry (mean fluorescence index 17314±2370 vs. 11930±2530, P=0.002 in bone marrow basophilic erythroblasts) are increased in β-thalassemic (th1/th1) relative to wild type (WT) mice. We hypothesized that a relative decrease in TfR1 expression would improve the phenotype in β-thalassemic mice and crossed TfR1+/- (TfR1 heterozygote) mice [Levy JE Nat Gen 1999] with th3/+ mice, another commonly used mouse model of β-thalassemia. Of the 50 pups born, 13 had th3 genotype, 12 (92%) of which also contained the mutant TfR1, suggesting a strong survival advantage of TfR1 heterozygote th3/+ (compound heterozygotes) relative to th3/+ mice. Analysis of 3-4 month old compound heterozygotes revealed a significant decrease in splenomegaly (0.007±0.001 vs. 0.016±0.0041 g spleen/g body weight, P=0.0009), reticulocytosis (1019±186 vs. 1672±218 x 10^9 cells/uL, P=0.001), and α-globin precipitation on circulating RBCs (Figure 1) relative to th3/+ mice. Furthermore, compound heterozygotes exhibit improvement in circulating RBCs (12±0.1 vs. 9±0.6 x 10^6 cells/uL, P We previously demonstrate that exogenous apo-transferrin (apoTf) injections result in more circulating RBCs, increased hemoglobin, and reversal of splenomegaly in th1/th1 mice [Li H Nat Med 2010]. We hypothesize that ineffective erythropoiesis in th1/th1 mice is TfR1-mediated and involves excess iron delivery to erythroid precursors. To further explore the role of TfR1 in erythropoiesis, we evaluate apoTf-treated th1/th1 mice. TfR1 mRNA expression is unchanged in apoTf-treated relative to untreated th1/th1 mice despite more iron restricted erythropoiesis (MCH 24.56±0.72 vs. 33.98±1.67 pg, P Taken together, our data suggest that TfR1 plays a critical role in erythropoiesis, both in an iron-dependent and possibly independent capacity. We postulate that a defect in TfR1 trafficking, perhaps with a delayed or incomplete removal of TfR1 during erythroid differentiation, occurs in β-thalassemia, that reduction of TfR1 in β-thalassemic mice partially reverses ineffective erythropoiesis, and that exogenous apoTf decreases TfR1 expression and exosomal release while increasing membrane and endosomal cycling. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Apoptosis, which leads to phagocytosis by mononuclear cells, represents the primary mechanism for removing neutrophils from inflamed tissues and minimizing injury. The present studies show that membrane phosphatidylserine turnover and permeability, as well as DNA fragmentation, were reduced in neutrophils from neonates when compared with adults. The activity of caspase 3 and expression of the proapoptotic proteins Bax, Bad, and Bak were also decreased in neonatal relative to adult neutrophils. These findings are consistent with impaired apoptosis in neonatal cells, which may contribute to prolonged inflammation in infants after oxidative stress or infection. Neutrophil apoptosis is induced by endogenous ligands such as Fas (FasL), which engage death receptors of the tumor necrosis factor/nerve growth factor superfamily, including Fas receptor (FasR). We found that expression of FasR was decreased in neonatal when compared with adult cells. Moreover, neonatal neutrophils did not undergo apoptosis in response to anti-FasR antibody and exhibited impaired chemotaxis to soluble FasL. However, in both adult and neonatal cells, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase inhibitors blocked Fas-induced activity. These data suggest that prolonged survival of neonatal neutrophils at injured sites is due, in part, to reduced responsiveness to FasL. This may be related to decreased expression of both FasR and Bcl-2–family proteins that mediate neutrophil apoptosis.