Although red blood cell (RBC) transfusions can be lifesaving, they are not without risk. In critically ill patients, RBC transfusions are associated with increased morbidity and mortality, which may increase with prolonged RBC storage before transfusion. The mechanisms responsible remain unknown. We hypothesized that acute clearance of a subset of damaged, stored RBCs delivers large amounts of iron to the monocyte/macrophage system, inducing inflammation. To test this in a well-controlled setting, we used a murine RBC storage and transfusion model to show that the transfusion of stored RBCs, or washed stored RBCs, increases plasma nontransferrin bound iron (NTBI), produces acute tissue iron deposition, and initiates inflammation. In contrast, the transfusion of fresh RBCs, or the infusion of stored RBC-derived supernatant, ghosts, or stroma-free lysate, does not produce these effects. Furthermore, the insult induced by transfusion of stored RBC synergizes with subclinical endotoxinemia producing clinically overt signs and symptoms. The increased plasma NTBI also enhances bacterial growth in vitro. Taken together, these results suggest that, in a mouse model, the cellular component of leukoreduced, stored RBC units contributes to the harmful effects of RBC transfusion that occur after prolonged storage. Nonetheless, these findings must be confirmed by prospective human studies.

Transfusions of RBCs stored for longer durations are associated with adverse effects in hospitalized patients. We prospectively studied 14 healthy human volunteers who donated standard leukoreduced, double RBC units. One unit was autologously transfused "fresh" (3-7 days of storage), and the other "older" unit was transfused after 40 to 42 days of storage. Of the routine laboratory parameters measured at defined times surrounding transfusion, significant differences between fresh and older transfusions were only observed in iron parameters and markers of extravascular hemolysis. IntroductionThe safety of transfusing RBCs after longer durations of refrigerated storage was recently identified as "the most critical issue facing transfusion medicine." 1 page 667 Concern was heightened when a large observational study of cardiac surgery patients found an increased risk of postoperative complications and reduced survival in those who received RBCs stored for more than 14 days. 2 Although still controversial, adverse clinical consequences have since been reported in most, [3][4][5] although not all, 6,7 epidemiologic studies of transfusions of RBCs stored for longer durations, but still within Food and Drug Administration (FDA) guidelines. The association between the duration of RBC storage and increased rates of serious infections, sepsis, and mortality is particularly strong in trauma patients. [7][8][9][10][11] Definitive determination of the potential risks associated with transfusion of RBCs stored for longer durations has been elusive, in part because the mechanisms responsible have not yet been identified.More than 14 million RBC units are transfused in the United States each year, with a mean storage interval of 18 days before transfusion. 12 During storage, RBCs undergo cumulative biochemical and biomechanical changes (the "storage lesion") that reduce their survival in vivo after transfusion. 13,14 In mouse models, 15 transfusion of RBCs stored for longer durations was followed by brisk extravascular clearance of a subpopulation of these cells, which were damaged during storage and removed by macrophages in the spleen and liver of recipient mice. The iron liberated by phagocytic digestion of these RBCs rapidly entered the systemic circulation in amounts that exceeded the transport capacity of plasma transferrin, the physiologic iron-binding protein; in this way, circulating non-transferrin-bound iron appeared and promoted the proliferation of pathogenic bacteria both in vitro 15 and in vivo. 16 We hypothesized that the infectious complications observed in human patients after transfusion of RBCs stored for longer durations were, at least in part, the result of the production of circulating non-transferrin-bound iron. Therefore, we prospectively examined healthy human volunteers to determine (1) if transfusion of autologous RBCs stored for longer durations was followed by the appearance of circulating non-transferrin-bound iron in vivo, and (2) if this increased circulating non-transferrinbound iron was assoc...

NIH grant HL115557 and UL1 TR000040.

Mutations in the recently identified presenilin 1 gene on chromosome 14 cause early onset familial Alzheimer disease (FAD). Herein we describe the expression and analysis of the protein coded by presenilin 1 (PS1) in NT2N neurons, a human neuronal model system. PS1 was expressed using recombinant Semliki Forest virions and detected by introduced antigenic tags or antisera to PS1-derived peptides. Immunoprecipitation revealed two major PS1 bands of approximately 43 and 50 kDa, neither of which were N-glycosylated or 0-glycosylated. Immunoreactive PS1 was detected in cell bodies and dendrites of NT2N neurons but not in axons or on the cell surface. PS1 was also detected in BHK cells, where it was also intracellular and colocalized with calnexin, a marker for the rough endoplasmic reticulum. A mutant form of PS1 linked to FAD did not differ from the wild-type protein at the light microscopic level. The model system described here will enable studies of the function of PS1 in human neurons and the role of mutant PS1 in FAD.Since mutations in the PS1 and PS2 genes result in early onset FAD that is quite similar to FAD in other pedigrees with APP mutations, the PS mutations may cause AD by altering the metabolism of APP and/or AP3, thereby accelerating amyloidogenesis. Considerable evidence suggests that neurons are a major source of the AP3 that forms amyloid deposits in AD (17, 18), and a recent study showed that within the brain PS1 and PS2 mRNAs are expressed primarily in neurons (19). For these reasons, it is important to study the biosynthesis and processing of these genes in neurons. To accomplish this goal, we developed a strategy to efficiently express and detect PS1 in a well characterized in vitro model of human neurons (NT2N cells) (20). We found that PS1 was localized in the cell body and dendrites of NT2N neurons but not on the cell surface or in axons.

The role of insulin (INS), and insulin-like growth factor-I (IGF-I Both INS and IGF-I activated signal transduction in maturing human erythropoietic cells as determined by phosphorylation of the insulin receptor substrate-2 (IRS-2) protein. We also found by RT-PCR that mRNA coding for INS-R is expressed in FACS sorted CD34؉ , c-kit-R ؉ marrow cells, and in cells isolated from BFU-E and CFU-GM colonies. Expression of INS-R protein on these cells was subsequently confirmed by cytofluorometry. In contrast, the receptor for insulin-like growth factor-I (IGF-IR) was not detected on CD34؉ cells, and was first easily detectable on more differentiated cells derived from day 6 BFU-E and CFU-GM colonies. We conclude that INS and IGF-I may be survival factors for human CD34 ؉ cells, but are not required during early erythropoiesis. In contrast, both growth factors may play some role at the final stages of erythroid maturation.