CUTE CHEST SYNDROME AND painful episodes are the most common precedents of death in adults with sickle cell anemia (SCA). 1 A randomized, doubleblinded, placebo-controlled trial, the

The binding of apotransferrin to the transferrin receptor on the surface of human leukemic K562 cells was found to be significantly less tight than that of the holoprotein, diferric transferrin. The finding that both ligands displayed linear Scatchard plots with similar receptor number ("'150,000 per cell) and mutually inhibit each other's binding suggested that they bind to the same receptor. Both the dissociation and association rate of apotransferrin were markedly increased (28-fold and 15-fold, respectively) at pH 7.2 compared to pH 4.8. Using the values of these binding parameters, we propose a mechanism to account for the recycling of transferrin subsequent to internalization and residence within an acidic nonlysosomal organelle where iron is removed.A wide variety of molecules gain entry into cells by receptormediated endocytosis (1,2). Diverse ligands, including low density lipoproteins, asialoglycoproteins, epidermal growth factor, a2 macroglobulin, lysosomal enzymes, and certain hormones, toxins, and viruses traverse strikingly similar, if not identical, pathways. Binding to specific cell-surface receptors is followed by internalization involving specialized regions of the plasma membrane, the coated pits. Between their internalization and their ultimate fate, which is often lysosomal degradation, appropriately tagged ligands are visible by electron microscopy within a number of morphologically varied intracellular vesicles. We have examined this pathway for the receptor-mediated endocytosis of asialoglycoproteins in hepatocytes and have shown that the receptor enters the cell as a complex with its ligand, whereupon the latter subsequently dissociates and is degraded (3, 4). In contrast to the ligand, the receptor is reutilized (5, 6). We have presented evidence that the intracellular dissociation of asialoglycoproteins is facilitated by encounter with an acidic environment prior to delivery to lysosomes (4, 7), a step that we believe to be critical in receptor reutilization. There is growing evidence that a number of other ligands are similarly exposed to nonlysosomal environments of low pH in their movement through the cell (8-11). This suggests that an acidic endocytic vesicle may participate in the intracellular transit of many ligands. As with asialoglycoproteins, a number of ligands interact with their respective receptors in a highly pH-dependent fashion, and many of these receptors have been shown to be reutilized (1).A notable exception to this generalized picture of receptormediated endocytosis involves the iron-binding protein transferrin (reviewed in ref. 12). Diferric transferrin enters the cell bound to its specific receptor, but internalization of the receptor-bound transferrin does not result in transferrin degradation (13)(14)(15). Instead, both the receptor and the apoprotein ligand are returned to the cell's exterior with retention of iron within the cell. Although coated pits (16) and an acidic, nonlysosomal microenvironment (9) have been shown to be involved in the transfe...

A randomized, controlled clinical trial established the efficacy and safety of short-term use of hydroxyurea in adult sickle cell anemia. To examine the risks and benefits of long-term hydroxyurea usage, patients in this trial were followed for 17.5 years during which they could start or stop hydroxyurea. The purpose of this follow-up was to search for adverse outcomes and estimate mortality. For each outcome and for mortality, exact 95% confidence intervals were calculated, or tests were conducted at a 5 0.05 level (P-value <0.05 for statistical significance). Although the death rate in the overall study cohort was high (43.1%; 4.4 per 100 person-years), mortality was reduced in individuals with long-term exposure to hydroxyurea. Survival curves demonstrated a significant reduction in deaths with long-term exposure. Twenty-four percent of deaths were due to pulmonary complications; 87.1% occurred in patients who never took hydroxyurea or took it for <5 years. Stroke, organ dysfunction, infection, and malignancy were similar in all groups. Our results, while no longer the product of a randomized study because of the ethical concerns of withholding an efficacious treatment, suggest that long-term use of hydroxyurea is safe and might decrease mortality. Am. J. Hematol. 85:403-408, 2010. V

At physiological temperature, the Fe-carrier transferrin is taken up by K562 human erythroleukemia cells through receptor-mediated endocytosis. Both ligand (now minus Fe) and receptor recycle back to the cell surface where the receptor is rapidly reutilized. After endocytosis, transferrin becomes transiently lodged within an acidic compartment inside the cell, as judged by the changed spectral characteristics and quantum yield of fluorescein isothiocyanate-labeled transferrin that is cell-associated at 37C. Upon binding to transferrin, anti-fluorescein antibody strongly quenches the emission of the fluoresceinlabeled residues on the protein and is used to assess whether the transferrin is at the cell surface (incubation at 0C) or mainly internalized into the cell (incubation at 37C). Using Percoll gradient fractionation of postnuclear supernatants, we show that the acidic compartment is not the lysosomal compartment.All cells require Fe, and the physiologic apparatus for Fe delivery utilizes a serum glycoprotein, transferrin (1). Despite some controversy concerning the pathway of Fe uptake, there is accumulating evidence pointing to receptor-mediated endocytosis of transferrin, with the subsequent transfer of Fe to the cell (2-5). There has been a great deal of interest in this general cellular process, and certain patterns are emerging from a number of endocytic systems (6). Such endocytosis is an energy-dependent process mediated by specific cell surface receptors, which are generally preferentially internalized through coated pits (7). Although the exact cellular path taken by the ligand is yet to be elucidated, most end up in lysosomes in which they are degraded (8, 9). The pathway taken by the receptor is even less clear. However, in several systems, the receptors clearly are not degraded and are reutilized (10, 11). We have studied certain details of the pathway taken by the asialoglycoprotein receptor (12). We showed that the ligand enters the cell attached to the receptor and that both receptor and ligand are internalized. Soon after internalization, the ligand is released from the receptor, and, after an obligate processing time, the ligand is degraded in the lysosomes. The receptor recycles back to the cell surface. The initial replacement for internalized receptor is derived from a cryptic spare receptor pool.In striking contrast to many other endocytosed ligands, transferrin is not degraded (3,13). Rather, mono-or diferric transferrin is taken up, the Fe removed, and the apotransferrin is released into the medium. Data from our laboratory (unpublished observations) concerning the transferrin uptake by K562 cells have led to the following conclusions. The K562 cells contain about 2 x 105 high-affinity (Kd = 1 x 10-9 M) transferrin surface-receptors per cell. Transferrin containing one or two ferric ions binds to these receptors and, when the cells are warmed, is rapidly internalized (t412 = 3 min). Upon internalization of the receptor-transferrin complex, the Fe is removed from the transferri...

This study evaluated efficacy and safety of darbepoetin alfa administered every 3 weeks (Q3W) at fixed doses of 300 or 500 μg with or without intravenous (IV) iron in treating anemia in patients receiving multicycle chemotherapy. This Phase 2, double‐blind, 2 × 2 factorial study randomized patients to one of four treatment arms; darbepoetin alfa 300 μg (n = 62), darbepoetin alfa 300 μg plus IV iron (n = 60), darbepoetin alfa 500 μg (n = 60), or darbepoetin alfa 500 μg plus IV iron (n = 60). Patients had nonmyeloid malignancies, hemoglobin levels ≤10 g dL−1, and no iron deficiency. Primary endpoint was achievement of target hemoglobin (≥11 g dL−1). Secondary endpoints included incidence of transfusions and change in Functional Assessment of Cancer Therapy Fatigue (FACT‐F) score from baseline to end of study. Safety was evaluated by incidence of adverse events. No evidence of a statistically significant interaction between darbepoetin alfa dose received and IV iron usage was observed, therefore, results are provided separately comparing darbepoetin alfa doses and comparing IV iron usage groups. Similar proportions of patients receiving darbepoetin alfa 300 or 500 μg achieved target hemoglobin (75 and 78%, respectively); Kaplan–Meier median time to target hemoglobin was 10 and 8 weeks, respectively. More patients receiving IV iron (82%) than not receiving IV iron (72%) achieved hemoglobin target. Adverse events profiles were similar for darbepoetin alfa treatment groups. Transient anaphylactoid reactions were reported in two patients receiving IV iron. Darbepoetin alfa at 300 μg Q3W and 500 μg Q3W showed similar benefit, while added IV iron improved treatment response in these patients. Am. J. Hematol., 2010. © 2010 Wiley‐Liss, Inc.