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Blood storage promotes the rapid depletion of red blood cell (RBC) high-energy adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG), which are critical regulators of erythrocyte physiology and function, as well as oxygen kinetics and posttransfusion survival. Sphingosine-1-phosphate (S1P) promotes fluxes through glycolysis. We hypothesized that S1P supplementation to stored RBC units would improve energy metabolism and posttransfusion recovery. We quantified S1P in 1929 samples (n = 643, storage days 10, 23, and 42) from the REDS RBC Omics study. We then supplemented human and murine RBCs from good storer (C57BL6/J) and poor storer strains (FVB) with S1P (1, 5, and 10 μM) before measurements of metabolism and posttransfusion recovery. Similar experiments were repeated for mice with genetic ablation of the S1P biosynthetic pathway (sphingosine kinase 1 [Sphk1] knockout [KO]). Sample analyses included metabolomics at steady state, tracing experiments with 1,2,3-13C3-glucose, proteomics, and analysis of end-of-storage posttransfusion recovery, under normoxic and hypoxic storage conditions. Storage promoted decreases in S1P levels, which were the highest in units donated by female or older donors. Supplementation of S1P to human and murine RBCs boosted the steady-state levels of glycolytic metabolites and glycolytic fluxes, ie the generation of ATP and DPG, at the expense of the pentose phosphate pathway. Lower posttransfusion recovery was observed upon S1P supplementation. All these phenomena were reversed in Sphk1 KO mice or with hypoxic storage. S1P is a positive regulator of energy metabolism and a negative regulator of antioxidant metabolism in stored RBCs, resulting in lower posttransfusion recoveries in murine models.

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, in reactions to transfusions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. Harmful antibodies often activate the complement cascade. A model for how IgG antibodies trigger complement activation involves interactions between IgG Fc domains driving the assembly of IgG hexamer structures that activate C1 complexes. The importance of IgG hexamers in initiating injury responses was not clear, so we tested their relevance in a mouse model of alloantibody- and complement-mediated acute lung injury. We used 3 approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer 'decoy' therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate an in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases., Introduction Antibodies and the complement cascade mediate protective immunity but can both become misdirected to cause harm in autoimmune and alloimmune diseases. Some antibodies direct activation of the complement cascade [...]

Administration of anti-RhD immunoglobulin (Ig) to decrease maternal alloimmunization (antibody-mediated immune suppression [AMIS]) was a landmark clinical development. However, IgG has potent immune-stimulatory effects in other settings (antibody-mediated immune enhancement [AMIE]). The dominant thinking has been that IgG causes AMIS for antigens on RBCs but AMIE for soluble antigens. However, we have recently reported that IgG against RBC antigens can cause either AMIS or AMIE as a function of an IgG subclass. Recent advances in mechanistic understanding have demonstrated that RBC alloimmunization requires the IFN-[alpha]/-[beta] receptor (IFNAR) and is inhibited by the complement C3 protein. Here, we demonstrate the opposite for AMIE of an RBC alloantigen (IFNAR is not required and C3 enhances). RBC clearance, C3 deposition, and antigen modulation all preceded AMIE, and both [CD4.sup.+] T cells and marginal zone B cells were required. We detected no significant increase in antigen-specific germinal center B cells, consistent with other studies of RBC alloimmunization that show extrafollicular-like responses. To the best of our knowledge, these findings provide the first evidence of an RBC alloimmunization pathway which is IFNAR independent and C3 dependent, thus further advancing our understanding of RBCs as an immunogen and AMIE as a phenomenon., Introduction Immunoglobulins are most commonly thought of as immune effector molecules. However, it has been known for over a century that Igs can also substantially enhance primary humoral immunity to [...]

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures ("exposome") may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell-Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine - though rarely detected in the blood donor population - has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could - through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin - improve erythrocyte metabolism and storage quality.

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.

BackgroundCigarette smoking is a frequent habit across blood donors (approx. 13% of the donor population), that could compound biologic factors and exacerbate oxidant stress to stored red blood cells (RBCs).Study design and methodsAs part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, a total of 599 samples were sterilely drawn from RBC units stored under blood bank conditions at Storage Days 10, 23, and 42 days, before testing for hemolysis parameters and metabolomics. Quantitative measurements of nicotine and its metabolites cotinine and cotinine oxide were performed against deuterium-labeled internal standards.ResultsDonors whose blood cotinine levels exceeded 10 ng/mL (14% of the tested donors) were characterized by higher levels of early glycolytic intermediates, pentose phosphate pathway metabolites, and pyruvate-to-lactate ratios, all markers of increased basal oxidant stress. Consistently, increased glutathionylation of oxidized triose sugars and lipid aldehydes was observed in RBCs donated by nicotine-exposed donors, which were also characterized by increased fatty acid desaturation, purine salvage, and methionine oxidation and consumption via pathways involved in oxidative stress-triggered protein damage-repair mechanisms.ConclusionRBCs from donors with high levels of nicotine exposure are characterized by increases in basal oxidant stress and decreases in osmotic hemolysis. These findings indicate the need for future clinical studies aimed at addressing the impact of smoking and other sources of nicotine (e.g., nicotine patches, snuff, vaping, secondhand tobacco smoke) on RBC storage quality and transfusion efficacy.

The US Food and Drug Administration (FDA) held a workshop on red blood cell (RBC) product regulatory science on October 6 and 7, 2016, at the Natcher Conference Center on the National Institutes of Health (NIH) Campus in Bethesda, Maryland. The workshop was supported by the National Heart, Lung, and Blood Institute, NIH; the Department of Defense; the Office of the Assistant Secretary for Health, Department of Health and Human Services; and the Center for Biologics Evaluation and Research, FDA. The workshop reviewed the status and scientific basis of the current regulatory framework and the available scientific tools to expand it to evaluate innovative and future RBC transfusion products. A full record of the proceedings is available on the FDA website (http://www.fda.gov/BiologicsBloodVaccines/NewsEvents/WorkshopsMeetingsConferences/ucm507890.htm). The contents of the summary are the authors' opinions and do not represent agency policy.

Oxidative stress can damage tissues and cells, and their resilience or susceptibility depends on the robustness of their antioxidant mechanisms. The latter include small molecules, proteins, and enzymes, which are linked together in metabolic pathways. Red blood cells are particularly susceptible to oxidative stress due to their large number of hemoglobin molecules, which can undergo auto-oxidation. This yields reactive oxygen species that participate in Fenton chemistry, ultimately damaging their membranes and cytosolic constituents. Fortunately, red blood cells contain robust antioxidant systems to enable them to circulate and perform their physiological functions, particularly delivering oxygen and removing carbon dioxide. Nonetheless, if red blood cells have insufficient antioxidant reserves (e.g., due to genetics, diet, disease, or toxin exposure), this can induce hemolysis in vivo or enhance susceptibility to a "storage lesion" in vitro, when blood donations are refrigerator-stored for transfusion purposes. Ergothioneine, a small molecule not synthesized by mammals, is obtained only through the diet. It is absorbed from the gut and enters cells using a highly specific transporter (i.e., SLC22A4). Certain cells and tissues, particularly red blood cells, contain high ergothioneine levels. Although no deficiency-related disease has been identified, evidence suggests ergothioneine may be a beneficial "nutraceutical." Given the requirements of red blood cells to resist oxidative stress and their high ergothioneine content, this review discusses ergothioneine's potential importance in protecting these cells and identifies knowledge gaps regarding its relevance in enhancing red blood cell circulatory, storage, and transfusion quality. [ABSTRACT FROM AUTHOR]

BACKGROUND. Glucose-6-phosphate dehydrogenase (G6PD) deficiency decreases the ability of red blood cells (RBCs) to withstand oxidative stress. Refrigerated storage of RBCs induces oxidative stress. We hypothesized that G6PD-deficient donor RBCs would have inferior storage quality for transfusion as compared with G6PD-normal RBCs. METHODS. Male volunteers were screened for G6PD deficiency; 27 control and 10 G6PD-deficient volunteers each donated 1 RBC unit. After 42 days of refrigerated storage, autologous 51-chromium 24-hour posttransfusion RBC recovery (PTR) studies were performed. Metabolomics analyses of these RBC units were also performed. RESULTS. The mean 24-hour PTR for G6PD-deficient subjects was 78.5% [+ or -] 8.4% (mean [+ or -] SD), which was significantly lower than that for G6PD-normal RBCs (85.3% [+ or -] 3.2%; P = 0.0009). None of the G6PD-normal volunteers (0/27) and 3 G6PD-deficient volunteers (3/10) had PTR results below 75%, a key FDA acceptability criterion for stored donor RBCs. As expected, fresh G6PD-deficient RBCs demonstrated defects in the oxidative phase of the pentose phosphate pathway. During refrigerated storage, G6PD-deficient RBCs demonstrated increased glycolysis, impaired glutathione homeostasis, and increased purine oxidation, as compared with G6PD-normal RBCs. In addition, there were significant correlations between PTR and specific metabolites in these pathways. CONCLUSION. Based on current FDA criteria, RBCs from G6PD-deficient donors would not meet the requirements for storage quality. Metabolomics assessment identified markers of PTR and G6PD deficiency (e.g., pyruvate/lactate ratios), along with potential compensatory pathways that could be leveraged to ameliorate the metabolic needs of G6PD-deficient RBCs. TRIAL REGISTRATION. ClinicalTrials.gov NCT04081272. FUNDING. The Harold Amos Medical Faculty Development Program, Robert Wood Johnson Foundation grant 71590, the National Blood Foundation, NIH grant UL1 TR000040, the Webb-Waring Early Career Award 2017 by the Boettcher Foundation, and National Heart, Lung, and Blood Institute grants R01HL14644 and R01HL148151., Introduction Red blood cell (RBC) transfusion is one of the most common therapeutic interventions for hospitalized patients (1). In particular, chronic transfusions are essential for patients with sickle cell disease [...]

Red blood cells (RBC) are the most abundant cell in the human body, with a central role in oxygen transport and its delivery to tissues. However, omics technologies recently revealed the unanticipated complexity of the RBC proteome and metabolome, paving the way for a reinterpretation of the mechanisms by which RBC metabolism regulates systems biology beyond oxygen transport. The new data and analytical tools also informed the dissection of the changes that RBCs undergo during refrigerated storage under blood bank conditions, a logistic necessity that makes >100 million units available for life-saving transfusions every year worldwide. In this narrative review, we summarize the last decade of advances in the field of RBC metabolism in vivo and in the blood bank in vitro, a narrative largely influenced by the authors' own journeys in this field. We hope that this review will stimulate further research in this interesting and medically important area or, at least, serve as a testament to our fascination with this simple, yet complex, cell. [ABSTRACT FROM AUTHOR]

Passive immunization with anti-D can prevent maternal alloimmunization to RhD thereby preventing hemolytic disease of the fetus and newborn. Unexpectedly, anti-D fails in some cases and some monoclonal anti-D preparations paradoxically enhances alloimmunization. The underlying mechanisms modulating humoral alloimmunization by anti-D are unknown. We previously reported that IgG antibody subclasses differentially regulate alloimmunity in response to red blood cell (RBC) transfusions in a mousemodel; in particular, IgG2c significantly enhanced RBC alloantibody responses. Initial mechanistic studies revealed that IgG2c:RBC immune complexes were preferentially consumed by the splenic dendritic cell (DC) subsets that play a role in RBC alloimmunization. The deletion of activating Fcgamma receptors (FcgRs) (i.e., FcgRI, FcgRIII, and FcgRIV) on DCs abrogated IgG2cmediated enhanced alloimmunization. Because DCs express high levels of FcgRIV, which has high affinity for the IgG2c subclass, we hypothesized that FcgRIV was required for enhanced alloimmunization. To test this hypothesis, knockout mice and blocking antibodies were used to manipulate FcgR expression. The data presented herein demonstrate that FcgRIV, but not FcgRI or FcgRIII, is required for IgG2c-mediated enhancement of RBC alloantibody production. Additionally, FcgRI is alone sufficient for IgG2c-mediated RBC clearance but not for increased alloimmunization, demonstrating that RBC clearance can occur without inducing alloimmunization. Together, these data, combined with prior observations, support the hypothesis that passive immunization with an RBC-specific IgG2c antibody increases RBC alloantibody production through FcgRIV ligation on splenic conventional DCs (cDCs). This raises the question of whether standardizing antibody subclasses in immunoprophylaxis preparations is desirable and suggests which subclasses may be optimal for generating monoclonal anti-D therapeutics. [ABSTRACT FROM AUTHOR]

Bone marrow transplantation (BMT) represents a cure for nonmalignant hematological disorders. However, compared with the stringent conditioning regimens used when performing BMT to treat hematological malignancies, the reduced intensity conditioning regimen used in the context of nonmalignant hematological disorders leads to substantially higher rates of BMT rejection, presumably due to an intact immune system. The relevant patient population typically receives transfusion support, often including platelets, and the frequency of BMT rejection correlates with the frequency of transfusion. Here, we demonstrate that immunity to transfused platelets contributes to subsequent BMT rejection in mice, even when the BMT donor and recipient are MHC matched. We used MHC-matched bone marrow because, although immunity to transfused platelets is best characterized in relation to HLA-specific antibodies, such antibodies are unlikely to play a role in clinical BMT rejection that is HLA matched. However, bone marrow is not matched in the clinic for minor histocompatibility antigens, such as those carried by platelets, and we report that transfusion of minor histocompatibility antigen-mismatched platelets induced subsequent BMT rejection. These findings indicate previously unappreciated sequelae of immunity to platelets in the context of transplantation and suggest that strategies to account for minor histocompatibility mismatching may help to reduce the chance of BMT rejection in human patients., Introduction Patients who have bone marrow failure syndromes are typically given supportive care, including transfusions of rbc and platelets (PLTs). Some marrow failure patients subsequently undergo bone marrow transplantation (BMT) [...]