Proteomic analysis of the supernatant of red blood cell units: the effects of storage and leucoreduction.

BACKGROUND: Red blood cell (RBC) transfusion is a life-saving intervention for critically ill patients; however, it has been linked to increased morbidity and mortality. We hypothesize that a number of important proteins accumulate during routine storage of RBCs, which may explain some of the adverse effects seen in transfused patients. STUDY DESIGN: Five RBC units were drawn and divided (half prestorage leucoreduced (LR-RBC) and half left as an unmodified control (RBC). The supernatant was separated on days 1 and 42 of storage and proteomic analyses completed with in-gel tryptic digestion and nano-liquid chromatography tandem mass spectrometry. RESULTS: In RBC supernatants, 401 proteins were identified: 203 increased with storage, 114 decreased, and 84 were unchanged. In LR-RBC supernatant, 231 proteins were identified: 84 increased with storage, 30 decreased, and 117 were unchanged. Prestorage leucoreduction removed many platelet- and leucocyte-derived structural proteins; however, a number of intracellular proteins accumulated including peroxiredoxins (Prdx) 6 and latexin. The increases were confirmed by immunoblotting, including the T-phosphorylation of Prdx-6, indicating that it may be functioning as an active phospholipase. Active matrix metalloproteinase-9 also increased with a coinciding decrease in the metalloproteinase inhibitor 1 and cystatin C. CONCLUSION: We conclude that a number of proteins increase with RBC storage, which is partially ameliorated with leucoreduction, and transfusion of stored RBCs may introduce mediators that result in adverse events in the transfused host.

Hypotension and acute pulmonary insufficiency following transfusion of autologous red blood cells during surgery: a case report and review of the literature.

Transfusion of autologous blood is associated with fewer complications, although all untoward events of transfusion may not be negated with this strategy. We report a case of acute pulmonary insufficiency and hypotension following transfusion of autologous packed red blood cells (PRBCs) in a patient, who was undergoing major surgery. Anti-HLA class-I and class-II and anti-granulocyte antibodies were measured in the unit and in the recipient. Neutrophil (PMN)-priming activity was measured as the augmentation of the formyl-Met-Leu-Phe-activated respiratory burst. No immunoglobulins were identified; however, significant lipid-priming activity was present in the implicated, autologous PRBC unit that primed PMNs from both healthy people and the recipient. In addition, lipids, identical to those that accumulate during PRBC storage, caused significant hypotension when infused into rats at similar concentrations found in stored PRBCs. We conclude that the observed transfusion-related acute lung injury reaction with significant hypotension may be the result of two independent events: the first is related to inherent host factors, in this case major surgery, and the second is the infusion of lipids that accumulate during the routine storage of PRBCs.

Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.

Rho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G-->A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways. (Blood. 2000;96:1646-1654)

In vitro release of vascular endothelial growth factor during platelet aggregation.

Platelet aggregation is a cardinal feature of both vascular repair and vascular disease. During aggregation platelets release a variety of vasoactive substances; some of these promote angiogenesis, endothelial permeability, and endothelial growth, actions shared by vascular endothelial growth factor (VEGF). This study was undertaken to investigate the hypothesis that VEGF is released by aggregating platelets. We found that VEGF was secreted during the in vitro aggregation of platelet-rich plasma induced by thrombin, collagen, epinephrine, and ADP (range 23-518 pg VEGF/ml). Furthermore, serum VEGF levels were elevated compared with plasma (230 +/- 63 vs. 38 +/- 8 pg VEGF/ml), indicative of VEGF release during whole blood coagulation. Lysates of apheresed, leukocyte-poor platelet units contained significant amounts of VEGF (2.4 +/- 0.8 pg VEGF/mg protein). VEGF message and protein were also present in a megakaryocytic cell line (Dami cell). These results suggest constitutive roles for platelet VEGF in the repair of intimal vessel injury and in the altered permeability and intimal proliferation seen at sites of platelet aggregation and thrombosis.

The effects of stem cell factor and granulocyte colony stimulating factor therapy on the activity of the neutrophil NADPH oxidase enzyme system.

BACKGROUND: To explore the effect of cytokine therapy on the NADPH oxidase in mature myeloid cells, we isolated neutrophils from patients receiving recombinant human granulocyte colony stimulating factor (G-CSF) and recombinant human stem cell factor (SCF) and evaluated oxidase activity. All patients had relapsed neoplastic disease and were at least 3 three weeks since the last course of chemotherapy or cytokine therapy. METHODS: Stimulus induced superoxide anion (O2-) production in response to PMA (200 ng/mL), fMLP (1 mumol/L), platelet activating factor (PAF, 2 mumol/L) priming of the fMLP induced response, and opsonized zymosan OZ (1 mg/mL) was measured. Polymorphonuclear leukocyte (PMN) subcellular components were prepared, after nitrogen cavitation, by separation on discontinuous sucrose gradients and NADPH oxidase activity was assessed in a SDS cell-free system. RESULTS: SCF had no effect on the activity of the neutrophil oxidase. Neutrophils isolated from patients treated with G-CSF and stimulated with PMA produced less (superoxide anion) O2- after therapy. PAF priming of the fMLP induced respiratory burst was also reduced after therapy with G-CSF. Subcellular NADPH oxidase activity was reduced before cytokine therapy commenced. This activity did not improve with cytokine treatment. CONCLUSIONS: It appears likely from this study that G-CSF therapy, with or without SCF, does not cause significant enhancement of neutrophil NADPH oxidase activity.

The association of biologically active lipids with the development of transfusion-related acute lung injury: a retrospective study.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is clinically similar to the adult respiratory distress syndrome (ARDS) and has been linked to the transfusion of leukocyte antibodies in blood components. Animal model have implicated neutrophil (PMN)-priming agents in ARDS; however, two agents were required. Previous studies showed the generation of PMN-priming agents during blood storage. Thus the association of PMN-priming agents with TRALI was examined. STUDY DESIGN AND METHODS: Ten patients with TRALI and 10 with febrile or urticarial reactions (control group) were evaluated. The presence of PMN-priming activity was tested in the patients' pretransfusion and posttransfusion blood samples by incubating PMNs with these samples followed by activation of the respiratory burst. Plasma lipids were separated by normal-phase high-performance liquid chromatography (HPLC), and the priming activity was evaluated. The presence of leukocyte antibodies was determined in the blood donors and patients with TRALI. RESULTS: Significantly more PMN-priming activity was present in the posttransfusion sera (11.4 +/- 1.8 nmol superoxide anion/min, mean +/- SEM; n = 10) and plasma of patients with TRALI than in their pretransfusion sera (6.5 +/- 1.5: n = 10) or in the pretransfusion and posttransfusion sera (5.1 +/- 1.3, n = 10; and 4.5 +/- 1.4, n = 10, respectively) and from the controls (p < 0.05). HPLC separation of lipids demonstrated that three active species were present in the posttransfusion plasma samples of TRALI patients. All the patients with TRALI had underlying clinical factors, such as infection, cytokine administration, recent surgery, or massive transfusion, while only 2 of 10 control patients had these clinical conditions. None of the donors had significant titers of HLA or HLA-DR antibodies; however, 50 percent had weak positivity for granulocyte antibodies. CONCLUSION: TRALI is the result of two clinical events, the first being a predisposing clinical condition and the second being the transfusion of biologically active lipids in stored blood.

WEB2170, a specific platelet-activating factor antagonist, attenuates neutrophil priming by human serum after clinical burn injury: the 1991 Moyer Award.

Primed neutrophils may contribute to endothelial and end-organ damage after burn injury because of increased endothelial adherence and enhanced toxic oxygen metabolite generation in response to a "second insult" such as bacterial sepsis. The purposes of this study were to determine: (1) whether serum from patients with thermal injury causes priming of the neutrophil NADPH:O2 oxidoreductase, (2) whether time after burn (early vs late) influences neutrophil priming, and (3) whether priming could be attenuated by a specific platelet-activating factor antagonist, WEB2170. Normal human neutrophils were incubated with 10% sera that was obtained from healthy adult controls (normal human sera) and with 10% sera from patients with greater than 30% total body surface area burns, which was collected early (early postburn sera) (i.e., between 12 and 48 hours after burn) or late (late postburn sera) (5 to 15 days, after burn). Priming of the neutrophil oxidase was tested for by measurement of the generation of superoxide anion after a stimulus of 10(-6) mol/L formyl-methionine-leucine-phenylalanine (fMLP). In separate experiments, neutrophils were pretreated with WEB2170 before serum incubation and fMLP stimulation to block any priming that may be mediated by platelet-activating factor. All sera caused an increased rate of superoxide anion production in response to fMLP and thus "primed" the neutrophil NADPH:O2 oxidoreductase. Greater priming occurred after incubation with late postburn sera than with other sera. WEB2170 completely inhibited priming by normal human sera and early postburn sera and partially inhibited priming by late postburn sera.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron spin echo envelope modulation evidence for carbonate binding to iron(III) and copper(II) transferrin and lactoferrin.

Iron binding to transferrin and lactoferrin requires a synergistic anion, which is carbonate in vivo. The anion is thought to play a key role in iron binding and release. To understand better the iron-carbonate interaction, experiments were performed with iron(III) and copper(II) complexes of human milk lactoferrin and serum transferrin with carbon-13-labeled carbonate. Modulation frequencies were present in the Fourier transforms of two-pulse and three-pulse electron spin echo envelope modulation data for the Fe(III) and Cu(II) complexes, consistent with binding of carbonate to both metals. The metal-13C interaction was similar for the lactoferrin and transferrin complexes. Spin coupling to the nitrogen of a coordinated histidine imidazole was observed for both metals. Both the metal-nitrogen and the metal-carbon spin coupling constants were about a factor of 5 smaller for the iron complexes than for the copper complexes, which indicated substantial similarity in the metal-carbonate and metal-imidazole binding for the two metals.

Hyponatremia and seizures in young children given DDAVP.

Desmopressin (DDAVP), a synthetic vasopressin, temporarily corrects bleeding abnormalities associated with mild hemophilia A, von Willebrand disease, and disorders of platelet function. The side effects of DDAVP are considered benign although most of its use has been in adults and older children. We report four children under the age of 2 years who became hyponatremic after intravenous DDAVP administration (0.3 microgram/kg). Three of them developed grand mal seizures. A review of the literature and these cases indicate that associated risk factors for hyponatremia after DDAVP administration include stress, surgery, anesthesia and narcotics (endogenous release of antidiuretic hormone), vomiting (loss of Na+), liver disease (hindered metabolism of DDAVP), renal tubular acidosis (chronically low serum Na+), multiple doses of DDAVP, and overhydration with hyponatremic fluids. DDAVP is not a benign drug in this age group and shows a serious potential for hyponatremia and seizures. Fluid restriction, avoidance of hyponatremic solutions, and close monitoring of serum electrolytes and urine output for at least 15-20 hr after the administration of DDAVP, when used in children under the age of 2 years, is warranted.

Intranasal administration of deferoxamine to iron overloaded patients.

We examined the effect of intranasal administration of deferoxamine on iron excretion in seven patients with iron overload secondary to chronic transfusion therapy. Deferoxamine was administered in doses of 0.75 to 3.0 gm given over 12 hours in a variety of dosing schedules. There was a probable, though not significant, dose response relationship between the amount of iron excreted and the dose administered. The amount of iron excreted was 10%-15% of that obtained using the same dosage of deferoxamine given by the subcutaneous route over the same time period. Hourly administration was more effective than less frequent administration. Addition of taurodeoxycholate to deferoxamine did not increase its absorption as measured by the levels of iron excretion. Side effects were few and consisted mainly of mild nasal irritation and a bad taste in the mouth. Nasal administration of deferoxamine may be a useful adjunct to iron chelation in patients receiving chronic transfusion therapy, particularly in those who are noncompliant with parenteral means of administration.

Inhibition of colony-stimulating factor (CSF) production by postburn serum: negative feedback inhibition mediated by lactoferrin.

Fatal infections in severely burned patients are often preceded by a decline in the production of colony-stimulating factor (CSF) and the proliferation of granulocyte-macrophage stem cells (CFU-GM), and overwhelming sepsis is often associated with leukopenia. The underlying mechanisms accounting for these granulopoietic defects are poorly understood, but the fact that postburn serum has been shown to inhibit CSF production suggests that a humoral factor or factors may play a role. Previous work has demonstrated that plasma levels of lactoferrin (LF), a known inhibitor of CSF production, are elevated following burn injury. To determine if LF is responsible for serum-mediated inhibition of CSF production, serial plasma levels of LF were measured in 18 burn patients using an enzyme-linked immunoabsorbent assay (ELISA). LF was elevated within 24 hours of injury and was associated with an absolute granulocytosis which rapidly declined, reaching a nadir at postburn days 3 through 5. Postburn serum, especially when collected during the first 24 hours following burn injury, inhibited in vitro CSF production by normal human peripheral blood mononuclear cells. Pre-incubation of postburn serum with an LF antibody restored normal CSF production. These data suggest that LF may play an important role in the regulation of postburn granulopoiesis.

Pancytopenia in propionic acidemia: hematologic evaluation and studies of hematopoiesis in vitro.

This study investigated the hematologic abnormalities of an infant with propionic acidemia and reversible pancytopenia. Light and electron microscopy of her bone marrow revealed severely disturbed cellular morphology with trilineage dysmyelopoiesis, hemophagocytosis, and numerous multinucleated histiocytes and megakaryocytes. The effects of her serum and of organic acids associated with propionic acidemia were studied on hematopoiesis in vitro. Mouse erythroid (CFU-E) and granulocyte-monocyte colonies (CFU-GM) were assayed by fibrin clot technique; human CFU-GM were grown in agar culture. The infant's serum reduced mouse CFU-E and CFU-GM by 43 and 32%, respectively, compared with normal human sera, but had no effect on human CFU-GM in our culture system. Buffered propionic acid caused concentration-dependent inhibition of mouse CFU-E and human CFU-GM over a range reported in sera of acutely ill infants with propionic acidemia. Neither cell viability nor subsequent colony formation was diminished by preincubation of bone marrow cells with propionic acid for 48 h. The three other organic acids studied, tiglic acid, 3-OH propionate, and glycine, did not inhibit growth of mouse CFU-E, CFU-GM, or human CFU-GM, and glycine significantly enhanced formation of the latter. Evaluation of the infant's hematologic abnormalities suggests that inhibition of bone marrow proliferation and maturation and, perhaps, shortened red blood cell survival were responsible for her pancytopenia. The studies performed in vitro implicate propionic acid in this hematopoietic dysfunction.

Measurement of adenosine triphosphate and 2,3-diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy.

Conditions for blood storage are chosen to assure adequate levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). Because of the invasive nature of the techniques, biochemical assays are not routinely used to measure levels of these compounds in stored blood. However, 31P NMR spectroscopy measures phosphorylated intermediates in intact cells and could be used without disruption of the storage pack. We compared levels of ATP and 2,3-DPG measured by 31P spectroscopy and standard enzyme-linked biochemical assays in whole blood (WB) and packed red blood cells (PRBCs) at weekly intervals during a 35-day storage period. NMR demonstrated a marked decrease in 2,3-DPG and an increase in inorganic phosphate after the first week of storage. No significant differences in ATP concentrations were seen in WB during the storage period, but a significant decrease in ATP in PRBCs was documented. There was good agreement in levels of ATP and 2,3-DPG measured by NMR and biochemical techniques. 31P NMR spectroscopy is a noninvasive technique for measuring ATP and 2,3-DPG which has a potential use in quality assurance of stored blood.

Oxidative metabolism in cord blood monocytes and monocyte-derived macrophages.

Little is known about phagocytosis-associated oxidative metabolism in mononuclear phagocytes from the human neonate. We investigated this phenomenon in monocytes from the cord blood of term newborn infants by measuring generation of superoxide anion (O2-) and hydroxyl radical (X OH) after stimulation with opsonized zymosan or phorbol myristate acetate. Production of these microbicidal oxygen metabolites by monocytes from neonates and healthy adult volunteers was equivalent. When cultured in the presence of the macrophage activator lipopolysaccharide or muramyl dipeptide, monocytes from neonates and adults differentiated into cells with the appearance of macrophages and with an enhanced capacity to release O2- compared with cells cultured in the absence of an activator. Monocyte-derived macrophages from neonates produced only slightly less O2- than did adult cells. Thus, unlike the cord blood neutrophil, which exhibits abnormalities in oxidative metabolism, the cord blood mononuclear phagocyte has a respiratory burst that is quantitatively comparable to that of the adult cell.

Defective bactericidal activity and absence of specific granules in neutrophils from a patient with recurrent bacterial infections.

Recent studies have suggested that specific granules and/or their contents may have a role in neutrophil adherence, oxidative metabolism, and other aspects of cell function. In the current report, we studied neutrophil function in a 13-year-old female with recurrent pyogenic infections and absent specific granules previously documented by electron microscopy. Levels of cobalamin (vitamin B12)-binding protein and lactoferrin were markedly decreased in this patient's neutrophils. Bactericidal activity against Escherichia coli was decreased at 60 and 120 min (percentage organisms killed: patient neutrophils, 48 and 33%; control neutrophils, 90 and 99%, respectively). Defective killing of Staphylococcus aureus was also documented. Degranulation and adherence were normal. Levels of lactoferrin and cobalamin-binding protein were decreased in plasma but normal in saliva, indicating that the defect was specific for hematopoietic tissue. Superoxide anion production was normal in the patient, while hydroxyl radical generation was decreased in response to opsonized zymosan. The data support the concept that specific granules and their contents are important for oxidative metabolism and other neutrophil functions.

Effect of subcutaneous deferoxamine and oral vitamin C on iron excretion in congenital hypoplastic anemia and refractory anemia associated with the 5q-syndrome.

Chronic refractory anemia associated with congenital hypoplastic anemia (CHA, Blackfan-Diamond syndrome) and with the 5q-syndrome may require chronic transfusion therapy to sustain life. Hemosiderosis and death from chronic iron overload may result from such a program. The effect of subcutaneous (SC) deferoxamine (DF) and supplemental oral vitamin C (vit. C) on urinary iron excretion was studied in two patients with congenital hypoplastic anemia and one patient with 5q-syndrome. In the two patients with CHA, urinary iron excretion in response to DF given SC over 24 hours was comparable to the results following intravenous (I.V.) administration. Both of these cases had low levels of plasma ascorbate on initial evaluation and excreted more iron in response to two different doses of DF after they had received supplemental vit C and their stores were repleted. Significant iron excretion occurred in all three patients for 12 hours during the SC infusion of DF and for 12 hours after the end of the infusion. In all three patients, increasing the dose of DF up to 3-4 g given SC over 12 hours resulted in a linear increase in iron excretion. Once normal body stores of ascorbate were achieved by oral supplementation, increasing doses of vit C did not appear to cause a further increment in iron excretion. DF administered by a slow SC infusion appears to be an effective approach to iron overload in patients with refractory anemia and hemosiderosis secondary to chronic transfusions. Only small amounts of supplemental vit. C necessary to sustain adequate body stores are required for optimal iron excretion.

Successful treatment of lymphohistiocytic reticulosis with phagocytosis with epipodophyllotoxin VP 16-213.

Lymphohistiocytic reticulosis with phagocytosis is a rare, familial disorder affecting infants and children. It is characterized by fever, pancytopenia, hepatosplenomegaly, and a rapidly fatal course. Prior attempts to treat this disease have been unsuccessful. We describe two patients with lymphohistiocytic reticulosis with phagocytosis and hyperlipidemia. A sibling of one patient had died of the same disease. One patient also had abnormal lymphocyte response to mitogens. Both patients who were treated with epipodophyllotoxin VP 16-213 (VP-16) had remission of their disease and resolution of hyperlipidemia. VP-16 appears to be an effective agent for treating lymphohistiocytic reticulosis with phagocytosis.