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Severe congenital neutropenia type 4 (SCN-4) is an autosomal recessive condition in which mutations in the G6PC3 gene encoding for the catalytic 3 subunit of glucose-6-phosphatase-β result in neutropenia, neutrophil dysfunction, and other syndromic features. We report a child with SCN-4 caused by compound heterozygous mutations in G6PC3, a previously identified missense mutation in exon 6 (c.758G>A[p.R235H]), and a novel missense mutation in exon 2 (c.325G>A[p.G109S]). The patient had recurrent bacterial infections, inflammatory bowel disease, neutropenia, and intermittent thrombocytopenia. Administration of granulocyte colony–stimulating factor (G-CSF) resolved the neutropenia and allowed for detailed evaluation of human neutrophil function. Random and directed migration by the patient’s neutrophils was severely diminished. Associated with this were defects in CD11b expression and F-actin assembly. Bactericidal activity at bacteria/neutrophil ratios >1:1 was also diminished and was associated with attenuated ingestion. Superoxide anion generation was

Nox1 is an abundant source of reactive oxygen species (ROS) in colon epithelium recently shown to function in wound healing and epithelial homeostasis. We identified Peroxiredoxin 6 (Prdx6) as a novel binding partner of Nox activator 1 (Noxa1) in yeast two-hybrid screening experiments using the Noxa1 SH3 domain as bait. Prdx6 is a unique member of the Prdx antioxidant enzyme family exhibiting both glutathione peroxidase and phospholipase A2 activities. We confirmed this interaction in cells overexpressing both proteins, showing Prdx6 binds to and stabilizes wild type Noxa1, but not the SH3 domain mutant form, Noxa1 W436R. We demonstrated in several cell models that Prdx6 knockdown suppresses Nox1 activity, whereas enhanced Prdx6 expression supports higher Nox1-derived superoxide production. Both peroxidase- and lipase-deficient mutant forms of Prdx6 (Prdx6 C47S and S32A, respectively) failed to bind to or stabilize Nox1 components or support Nox1-mediated superoxide generation. Furthermore, the transition-state substrate analogue inhibitor of Prdx6 phospholipase A2 activity (MJ-33) was shown to suppress Nox1 activity, suggesting Nox1 activity is regulated by the phospholipase activity of Prdx6. Finally, wild type Prdx6, but not lipase or peroxidase mutant forms, supports Nox1-mediated cell migration in the HCT-116 colon epithelial cell model of wound closure. These findings highlight a novel pathway in which this antioxidant enzyme positively regulates an oxidant-generating system to support cell migration and wound healing.

Variations in cord blood manufacturing and administration are common, and the optimal practice is not known. We compared processing and banking practices at 16 public cord blood banks (CBB) in the United States and assessed transplantation outcomes on 530 single umbilical cord blood (UCB) myeloablative transplantations for hematologic malignancies facilitated by these banks. UCB banking practices were separated into 3 mutually exclusive groups based on whether processing was automated or manual, units were plasma and red blood cell reduced, or buffy coat production method or plasma reduced. Compared with the automated processing system for units, the day 28 neutrophil recovery was significantly lower after transplantation of units that were manually processed and plasma reduced (red cell replete) (odds ratio, .19; P = .001) or plasma and red cell reduced (odds ratio, .54; P = .05). Day 100 survival did not differ by CBB. However, day 100 survival was better with units that were thawed with the dextran-albumin wash method compared with the "no wash" or "dilution only" techniques (odds ratio, 1.82; P = .04). In conclusion, CBB processing has no significant effect on early (day 100) survival despite differences in kinetics of neutrophil recovery.

There is concern at the National Heart, Lung, and Blood Institute (NHLBI) and among transfusion medicine specialists regarding the small number of investigators and studies in the field of pediatric transfusion medicine (PTM). Accordingly, the objective of this article is to provide a snapshot of the clinical and translational PTM research considered to be of high priority by pediatricians, neonatologists, and transfusion medicine specialists. Included is a targeted review of three research areas of importance: (i) transfusion strategies, (ii) short- and long-term clinical consequences, and (iii) transfusion-transmitted infectious diseases. The recommendations by PTM and transfusion medicine specialists represent opportunities and innovative strategies to execute translational research, observational studies, and clinical trials of high relevance to PTM. With the explosion of new biomedical knowledge and increasingly sophisticated methodologies over the past decade, this is an exciting time to consider transfusion medicine as a paradigm for addressing questions related to fields such as cell biology, immunology, neurodevelopment, outcomes research, and many others. Increased awareness of PTM as an important, fertile field and the promotion of accompanying opportunities will help establish PTM as a viable career option and advance basic and clinical investigation to improve the health and wellbeing of children.

Introduction: Chronic Granulomatous Disease (CGD) is a genetic disorder of the Nox2 enzyme system in phagocytic cells. Neutrophils and monocytes from patients with CGD are deficient in Nox2 components, unable to produce superoxide anion and other reactive oxygen species (ROS), and cannot kill certain types of bacteria and fungi. Patients with CGD suffer from recurrent, life-threatening infections. Treatment includes prophylaxis with antibiotic and antifungal agents and therapy with IFN-γ. Results from clinical trials of IFN-γ in patients with CGD have demonstrated decreased incidence and severity of infections. The exact mechanism(s) by which IFN-γ achieves its clinical effects is not clear. Previous studies from our laboratory in healthy adult volunteers demonstrated that administration of IFN-γ induced dramatic changes in the neutrophil phenotype with significant functional implications. To investigate IFN-γ induced changes in neutrophils further we initiated a study in CGD patients. Clinical Trial and Methods: Patients with CGD were enrolled on a study protocol approved by the COMIRB at the University of Colorado Denver. Patients between 5 and 60 years with defined genetic variants of CGD, but without recent infections or medical complications were entered onto the study. IFN-γ was stopped for a week to allow a drug washout period; prophylactic antibiotics and antifungal medications were continued. Blood samples were obtained from patients off IFN-γ, 12 hours after the first dose, and after the fourth dose of IFN-γ (50 mcg./m2 given on a standard schedule three times a week). Neutrophils were isolated by Dextran sedimentation, Ficoll Hypaque density centrifugation, and hypotonic lysis of red blood cells. RNA was isolated by standard technique and gene expression completed by RNAseq analysis (results pending). Ingestion was measured with fluorescently labeled S. aureus, bactericidal activity against S. aureus with 1:1 ratio of neutrophils to bacteria in 10% normal human serum by standard technique, and chemotaxis to fMLF and C5a with calcein-AM labeled neutrophils across fluorescence blocking PET membrane in Corning HTS Fluroblock plates. Superoxide anion was measured as SOD inhibitable cytochrome c reduction in response to standard agonists, CD11b expression and F-actin assembly in response to PMA and fMLF by flow cytometry, MHC Class II antigens by flow cytometry, nitric oxide (NO) levels in neutrophil lystes using a colorimetric assay to measure the combined nitrate and nitrite levels, and apoptosis determined as the combined caspase 3 and caspase 7 activity in isolated lysates using the Caspase-Glo 3/7 Assay from Promega. Results: We report here preliminary results on the first three patients studied; all are gp91phox deficient X-linked CGD. As expected, superoxide anion was not detected from patient neutrophils off INF-γ or during treatment. Ingestion and cell motility were not affected by IFN-γ administration. In all three subjects, caspase activity was decreased during IFN-γ treatment compared to before. In all three patients bactericidal activity against S. aureus was deficient; in 2/3 patients, administration of IFN was associated with a small but specific increase in killing, without change in ROS production. Strikingly, in two patients who showed improved bactericidal activity, NO production in lysates was increased after IFN-γ treatment compared to before and showed a further increase when cells were stimulated with fMLF. CD11b expression and F-actin assembly did not appear to be altered by IFN-γ treatment. Expression of HLA Class II markers (HLADRA and CD 274) appeared on neutrophils and increased in expression after initiation of IFN treatment. This change also was seen with CD 40. Summary: IFN-γ appeared to have dramatic effects on neutrophils from patients with CGD including possible improved bactericidal activity, the generation of NO which could be bactericidal and compensate for the deficiency of generation of ROS, and expression of MHC Class II antigens with possible association to antigen processing and enhanced interaction with adaptive immune function. Conclusion: Changes in the neutrophil phenotype induced by IFN-γ may provide alternative strategies compensating for the genetic deficiency of CGD. Defining how IFN-γ enhances neutrophil function may expand the use of this drug to other medical disorders in which innate immune response is involved. Disclosures Ambruso: Horizon Pharma Ireland Ltd.: Consultancy.

Interferon-gamma (IFN-γ) is a cytokine with powerful immunoregulatory effects. Most of these have been documented within the adaptive immune system and less has been reported about its effects on neutrophil function. Furthermore, most neutrophil studies have evaluated the effect of IFN- γ on mature cells isolated from peripheral blood thus potentially missing effects induced by its action on maturing neutrophils in the bone marrow. The clinical use of IFN-γ has been driven by these data and the clinical findings that administration of this cytokine to patients with Chronic Granulomatous Disease results in decreased incidence of severe infections without correcting the defect in phagocyte Nox2 activity. To determine the in vivo effects on neutrophils maturing in the marrow under the influence of this cytokine, we studied healthy human volunteers receiving IFN-γ at single escalating doses of 10, 25, 50, and 100 mcg/m2. Blood samples were obtained before and 4, 8, 12, 24, 36, 48, 72, and 96 hours after the administration. Plasma was stored for IFN-γ, IL-10 and neopterin determination by ELISA. Additionally, neutrophils were isolated from heparinized whole blood and superoxide anion generation after stimulation with PMA (200 ng/ml) and fMLF (1µM) was measured as SOD inhibitable cytochrome c reduction. RNA was isolated from neutrophils by standard techniques and genome wide changes in transcription were measured using Affymetrix gene chips. IFN-gamma concentrations in plasma spiked transiently after its administration and the magnitude and persistence of the resulting peaks were dose dependent. Levels of IL-10 and neopterin, two molecules known to respond to IFN-g, also exhibited time and dose dependent increases in plasma levels albeit with different kinetics; IL-10 responded with sharp peaks similar to the IFN- profiles, but neopterin showed slower developing and longer lasting increases. Superoxide anion generation by neutrophils in response to fMLF and PMA was quantitatively enhanced early after administration of IFN-γ returning to baseline by the end of the observation period. The increase in the respiratory burst exhibited a dose response with a plateau at 50 mcg/m2. Marked changes in gene expression were noted and, using the criteria of a 2-fold change over baseline, 866 genes showed increases and 1909 genes exhibited decreases in expression. The changes occurred rapidly after administration returning back towards baseline by 24-36 hours. Both dose dependent increases and decreases were observed. As expected, a number of genes directly associated with neutrophil function were increased such as Nox2 components (CYBB, NCF1), Fc receptors (FCGR1A, FCGR1B), and innate immune receptors including TLR5, TLR8 and LY96. Strikingly, dramatic changes were noted in the genes for proteins in MHCII and MCHI systems, guanylate binding proteins, and the chemokine receptor, CXCR4. In addition, the expression of GTP Cyclohydrolase 1 (GCH1), a rate limiting enzyme in the tetrahydrobiopterin (BH4) biosynthetic pathway, was increased 18-fold after IFN-γ administration. Since BH4 is a cofactor in nitric oxide (NO) synthases we speculated that increased GCH1 could enhance NO production in neutrophils. The upregulation of this pathway in neutrophils was confirmed with a demonstration that NO generation in neutrophil lysates spiked from low levels to peak 4-8 hours after IFN administration before returning toward baseline after 36 hours. The generation of NO as well as other changes in the neutrophil phenotype may provide compensatory strategies to afford more robust function to the cell limited by genetic defects of CGD or other neutrophil dysfunction disorders. These results demonstrate that administration of IFN-γ enhances classic neutrophil function and also induces previously under or unappreciated neutrophil functions. Under the influence of IFN-γ, the neutrophil alters its functional phenotype to include novel strategies that enhance its role in host defense and interaction with the adaptive immune system. Understanding these effects of IFN- γ will help define its clinical effects in CGD and extend its possible uses to other diseases. Disclosures Ambruso: Horizon Pharma Ireland Ltd: Other: Educational Consulting.

Although antibodies to antigens in the Rh blood group system are common causes of warm autoimmune hemolytic anemia, specificity for only the D antigen is rare in autoimmune hemolysis in pediatric patients. This case reports an anti-D associated with severe hemolytic anemia (Hb = 2.1 g/dL) in a previously healthy 14-month-old child who presented with a 3-day history of low-grade fevers and vomiting. Because of his severe anemia, on admission to the hospital he was found to have altered mental status, metabolic acidosis, abnormal liver function tests, and a severe coagulopathy. He was successfully resuscitated with uncrossmatched units of group O, D– blood, and after corticosteroid therapy he had complete resolution of his anti-D-mediated hemolysis. Immunohematology 2013;29:15–18.

Peroxiredoxin 6-phospholipase A2 (Prdx6-PLA2) is a bi-functional enzyme with peroxi-redoxin (Prdx) and phospholipase A2 (PLA2) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB-985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged. Reintroduction of shRNA-resistant Prdx6-PLA2 into the knockdown cells by stable transfection with a Prdx6-PLA2 expression plasmid restored the fMLF response, as did reintroduction of Prdx6-PLA2 mutated in the Prdx active site; reintroduction of PLA2 active site mutants, however, failed to restore the response. Thus, the PLA2 activity of Prdx6-PLA2 in intact cells mediates its ability to enhance phox activity in response to fMLF. In combination with previous publications by other groups, our work indicates that various PLA2 isoforms can enhance oxidase activity but they are differentially important in different cell types and in the response to different agonists.

Priming of polymorphonuclear leukocytes (PMNs) enhances their adhesion to endothelium, the release of their granule content and their production of reactive oxygen species. These effects are etiological in transfusion related acute lung injury (TRALI) and many clinically important mediators of TRALI prime PMNs. A priming activity that develops over time in stored blood products has been shown to be due to the accumulation of lysophospatidylcholines (lyso-PCs) and has been found to be related clinically to TRALI. Lyso- PCs prime PMNs activating the G2A receptor and several inhibitors of this receptor, which could potentially be therapeutic in TRALI, have been identified. Recent work has described early steps in the signaling from the G2A receptor which has revealed potential targets for novel antagonists of lyso-PC mediated priming via G2A. Additionally, characterization of the process by which lyso-PCs are generated in stored blood products could allow development of inhibitors and additive solutions to block their formation in the first place.

BACKGROUND: Pediatric transfusion medicine (PTM) is a subspecialty of transfusion medicine with no formal training program and few specialists. The Pediatric Transfusion Medicine Academic Awardees (PedsTMAA) group surveyed PTM content experts to identify relevant objectives for the first formal PTM curriculum. STUDY DESIGN AND METHODS: Eight North American PTM experts were invited to participate in a two-step consensus process. PTM-related objectives compiled from a review of existing training documents were organized into a survey. Experts were asked to rate each objective for relevancy for a clinical pathology trainee. Content validity indexes (CVIs) and asymmetric confidence intervals (ACIs) of expert ratings and analysis of respondents' comments were used to identify relevant objectives. RESULTS: Six experts participated and reviewed 117 objectives. Based on content validity criteria (CVI ≥ 0.83 and lower-limit 95% ACI ≥ 3), a total of 65 objectives were considered relevant. Twenty-three objectives were rated “very relevant” by all the experts while some proposed objectives were determined to be not relevant, out of date, or inappropriate for a resident trainee level. CONCLUSIONS: The PedsTMAA group identified 65 objectives for a PTM curriculum. Twenty-three represent a clear core set of objectives and should be considered for clinical pathology training. The next step is to consider the teaching strategies and evaluation methods that will be employed to best deliver this content addressing competency in medical knowledge.

Background: We investigated the clotting factor profile of cryoprecipitate produced from blood stored 24 hours (24hr CP) compared with standard cryoprecipitate (CP). Methods: Ten units of blood from healthy volunteers were collected and split. One set of products was processed at 8 hours for fresh frozen plasma (FFP) and cryoprecipitate (CP); the second group was processed at 24 hours to make frozen plasma (24hr FP) and 24hr CP. Activated partial thromboplastin time (aPTT), factor VIII activity, and antigen levels were completed on each sample. Cryoprecipitate was evaluated for fibrinogen, von Willebrand factor activity, factor VIII activity, and antigen levels. Results: Factor VIII activity declined by 29% in 24hr FP compared with FFP. Factor VIII activity, fibrinogen, and von Willebrand factor activity levels were not decreased in 24hr CP compared with CP. Conclusion: The 24hr CP met FDA and AABB standards for CP. The clinical efficacy and safety of 24hr CP should be confirmed.

Objective: The occurrence of non-hemolytic transfusion reactions is highest with platelet and plasma administration. Some of these reactions are characterized by endothelial leak, especially transfusion related acute lung injury (TRALI). Elevated concentrations of inflammatory mediators secreted by contaminating leukocytes during blood product storage may contribute to such reactions, but platelet-secreted mediators may also contribute. We hypothesized that platelet storage leads to accumulation of the endothelial permeability mediator vascular endothelial growth factor (VEGF), and that intravascular administration of exogenous VEGF leads to extensive binding to its lung receptors. Methods: Single donor, leukocyte-reduced apheresis platelet units were sampled over 5 days of storage. VEGF protein content of the centrifuged supernatant was determined by ELISA, and the potential contribution of VEGF from contaminating leukocytes was quantified. Isolated-perfused rat lungs were used to study the uptake of radiolabeled VEGF administered intravascularly, and the effect of unlabeled VEGF on lung leak. Results: There was a time-dependent release of VEGF into the plasma fraction of the platelet concentrates (62 ± 9 pg/ml on day one, 149 ± 23 pg/ml on day 5; mean ± SEM, p

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. Antibodies to HNA-3a are commonly implicated in TRALI. We hypothesized that HNA-3a antibodies prime neutrophils (PMNs) and cause PMN-mediated cytotoxicity through a two-event pathogenesis. Isolated HNA-3a+ or HNA-3a− PMNs were incubated with plasma containing HNA-3a antibodies implicated in TRALI, and their ability to prime the oxidase was measured. Human pulmonary microvascular endothelial cells (HMVECs) were activated with endotoxin or buffer, HNA-3a+ or HNA-3a− PMNs were added, and the coculture was incubated with plasma ± antibodies to HNA-3a. PMN-mediated damage was measured by counting viable HMVECs/mm2. Plasma containing HNA-3a antibodies primed the fMLP-activated respiratory burst of HNA-3a+, but not HNA-3a−, PMNs and elicited PMN-mediated damage of LPS-activated HMVECs when HNA-3a+, but not HNA-3a−, PMNs were used. Thus, antibodies to HNA-3a primed PMNs and caused PMN-mediated HMVEC cytotoxicity in a two-event model identical to biologic response modifiers implicated in TRALI.

Transfusion-related acute lung injury (TRALI) is a life-threatening adverse effect of transfusion that is occurring at increasing incidence in the United States and that, in the past 2 reporting years, has been the leading cause of transfusion-related death. TRALI and acute lung injury (ALI) share a common clinical definition except that TRALI is temporally and mechanistically related to the transfusion of blood/blood components. In prospective studies, 2 patient groups, 1 requiring cardiac surgery and 1 with hematologic malignancies and undergoing induction chemotherapy, were predisposed. Two different etiologies have been proposed. The first is a single antibody-mediated event involving the transfusion of anti-HLA class I and class II or antigranulocyte antibodies into patients whose leukocytes express the cognate antigens. The second is a 2-event model: the first event is the clinical condition of the patient resulting in pulmonary endothelial activation and neutrophil sequestration, and the second event is the transfusion of a biologic response modifier (including lipids or antibodies) that activates these adherent polymorphonuclear leukocytes (PMNs), resulting in endothelial damage, capillary leak, and TRALI. These hypotheses are discussed, as are the animal models and human studies that provide the experimental and clinical relevance. Prevention, treatment, and a proposed definition of TRALI, especially in the context of ALI, are also examined.

Rac2 is a Rho GTPase that is expressed in cells of hematopoietic origin, including neutrophils and macrophages. We recently described an immunodeficient patient with severe, recurrent bacterial infections that had a point mutation in one allele of the Rac2 gene, resulting in the substitution of aspartate 57 with asparagine. To ascertain further the effects of Rac2 D57N in leukocytes, Rac2 D57N was expressed in primary murine bone-marrow-derived macrophages (cells that we show express approximately equal amounts of Rac1 and Rac2). Rac2 D57N expression in macrophages inhibited membrane ruffling. Rac2 D57N expression inhibited the formation of macropinosomes, demonstrating a functional effect of the loss of surface membrane dynamics. Surprisingly, Rac2 D57N induced an elongated, spread morphology but did not affect microtubule networks. Rac2 D57N also inhibited lipopolysaccharide-stimulated p38 kinase activation. Examination of guanine nucleotide binding to recombinant Rac2 D57N revealed reduced dissociation of GDP and association of GTP. Coimmunoprecipitation studies of Rac2 D57N with RhoGDIα and Tiam1 demonstrated increased binding of Rac2 D57N to these upstream regulators of Rac signaling relative to the wild type. Enhanced binding of Rac2 D57N to its upstream regulators would inhibit Rac-dependent effects on actin cytoskeletal dynamics and p38 kinase signaling.

We read with interest the recent publication of Vlaar and colleagues,1 which detailed the accumulation of bio-active lipids during the storage of blood products and their findings that these lipids are not cell but plasma and temperature dependent. In this article the authors demonstrated that lyso-PCs did not accumulate during the routine storage of prestorage leukoreduced (buffy coat removal) red blood cells (LR-RBCs) in SAGM, 42 days at 2 to 4°C, and did not evidence priming activity using a 30-minute neutrophil (PMN) priming assay in which the respiratory burst was measured in the presence of the plasma or plasma fraction of the stored component. However, if plasma was added to the LR-RBCs then there was a significant increase in lyso-PCs on Day 1 that did not increase over the storage interval. In addition, platelet (PLT) concentrates, stored in plasma, did evidence lyso-PC accumulation during routine storage, 7 days at 20 to 24°C, and the plasma fraction primed the PMN oxidase after 30 minutes. The accumulation of lyso-PCs was inhibited; however, the priming activity was not abrogated when SSP was substituted as the storage solution (65%–95%)Final. The authors concluded that the observed accumulation of lyso-PCs was time and temperature dependent and had little to do with the cellular constituents of the stored product.

Production of toxic oxygen metabolites provides a mechanism for microbicidal activity of the neutrophil. The NADPH oxidase enzyme system initiates the production of oxygen metabolites by reducing oxygen to form superoxide anion (O(2)()). With stimulation of the respiratory burst, cytosolic oxidase components, p47(phox), p67(phox), and Rac, translocate to the phagolysomal and plasma membranes where they form a complex with cytochrome b(558) and express enzyme activity. A 29-kDa neutrophil protein (p29) was identified by co-immunoprecipitation with p67(phox). N-terminal sequence analysis of p29 revealed homology to an open reading frame gene described in a myeloid leukemia cell line. A cDNA for p29 identical to the open reading frame protein was amplified from RNA of neutrophils. Significant interaction between p29 and p67(phox) was demonstrated using a yeast two-hybrid system. A recombinant (rh) p29 was expressed in Sf9 cells resulting in a protein with an apparent molecular weight of 34,000. The rh-p29 showed immunoreactivity with the original rabbit antiserum that detected p47(phox) and p67(phox). In addition, rh-p29 exhibited PLA(2) activity, which was Ca(2+) independent, optimal at low pH, and preferential for phosphatidylcholine substrates. The recombinant protein protected glutathione synthetase and directly inactivated H(2)O(2). By activity and sequence homology, rh-p29 can be classified as a peroxiredoxin. Finally, O(2)() production by plasma membrane and recombinant cytosolic oxidase components in the SDS-activated, cell-free NADPH oxidase system were enhanced by rh-p29. This effect was not inhibited by PLA(2) inhibitors. Thus, p29 is a novel protein that associates with p67 and has peroxiredoxin activity. This protein has a potential role in protecting the NADPH oxidase by inactivating H(2)O(2) or altering signaling pathways affected by H(2)O(2).

The cytokine IFN-γ is used as a drug to treat chronic granulomatous disease (CGD), hyperimmunoglobulin E syndromes and osteopetrosis. The goal of this study was to better understand how IFN-γmight support PMN function in these diseases, and to obtain information that might expand potential uses for IFN-γ. Neutrophils mature in the bone marrow and then enter the blood where they quickly undergo apoptotic cell death; a range of human studies have estimated the neutrophil half-life at only 5-10 hours. Therefore we reasoned that IFN-γ might exert its effects on neutrophils via prolonged exposure to cells undergoing maturation toward a terminally differentiated state in the marrow rather than by its brief exposure to short-lived circulating cells. To explore this possibility we made use of PLB-985 cells which belong to a myeloblast-like myeloid cell line that can be differentiated into a mature, neutrophil-like state by treatment with various agents including DMSO. In initial studies we used microarrays and western blotting to compare transcription and protein expression in PLB-985 cells matured, using DMSO, in the presence or absence of IFN-γ. We observed changes in a significant number of mRNAs and proteins, that fall into the following categories, which could help explain the immune supportive effects of IFN-γ. 1 Genes known to be involved in classical aspects of neutrophil function, i.e. transmigration, chemotaxis, phagocytosis and pathogen killing 2 Genes involved in neutrophil clearance and homeostasis, including some involved in apoptosis. 3 Genes encoding innate immune receptors 4 Genes encoding guanylate binding proteins, a family of GTPases implicated in antimicrobial activity in different cell types but not, to our knowledge, neutrophils. These genes were among the most upregulated observed in this study, for example Gbp5 was increased 337 fold. 5 Genes involved in antigen presentation by MHCI and MHCII systems. Although neutrophils are generally considered to be involved in innate immunity a number of studies have suggested that they might act as antigen presenting cells and our finding of IFN-g induced upregulation of MHCII components is consistent with this. Some MHCI related genes were amongst the most upregulated genes in this study, for example HLA-DRA was increased 75 fold. Next we explored if the effect of IFN-γ on expression of these genes depends on whether the cells are undergoing maturation. This was done by comparing the effects of IFN-γ on maturing cells (i.e. those cultured in the presence of DMSO) versus non-maturing cells. To do this, the fold changes in mRNAs for DMSO plus IFN-γ treated cells versus DMSO treated cells were compared to the fold changes for IFN-γ treated cells versus untreated cells. In general, for genes showing the largest changes in mRNA expression in response IFN-γ, it was found that even larger changes occurred in maturing cells. This suggests that the effects of IFN-γon maturing cells are due to more than just prolonged application of the chemokine and are enhanced by ongoing developmental differentiation of the cells. Collectively the findings in this study enhance our understanding of the effects of IFN-γ on myeloid cells and indicate possible mechanisms by which this cytokine could support immune function. Disclosures No relevant conflicts of interest to declare.

Introduction: Most information about the immunoregulatory functions of INF-γ has focused on the interaction with the adaptive immune system and less is reported about neutrophil function. Furthermore, the majority of studies with neutrophils evaluate the effects of INF-γ on differentiated cells. The clinical use of IFN-γ has been driven by these data and the clinical findings that administration of this cytokine to patients with Chronic Granulomatous Disease results in decreased incidence of severe infections although the defect in Nox2 activity is not altered. To determine the in vivoeffects on myeloid cells developed under the influence of this cytokine, we evaluated neutrophils from healthy human subjects receiving IFN-γ. Methods: Healthy human volunteers were administered IFN-γ subcutaneously under an FDA approved IND and a protocol approved by the COMIRB at the University of Colorado. Healthy adults between 18 and 60 years of age with no history of recent infections were enrolled. IFN-γ, at single escalating doses of 10, 25, 50, and 100 mcg. /m2, was given subcutaneously at weekly intervals. Blood samples were obtained before and 4, 8, 12, 24, 36, 48, 72, and 96 hrs. after the administration. Plasma was stored for IL-10 and Neopterin levels assayed by ELISA techniques. Neutrophils were isolated from heparinized whole blood by Dextran sedimentation, Ficoll Hypaque density gradient centrifugation, and hypotonic lysis of red blood cells. Superoxide anion generation after stimulation with PMA (200 ng/ml) and fMLF (1µM) was measured as SOD inhibitable cytochrome c reduction. RNA was isolated by standard techniques. Analysis of gene expression was completed after preparation of labelled cDNA and hybridization with microarrays (Affimetrix GeneChip), normalization, transformation and assignment of relative expression levels. Results for the first 5 subjects are summarized here. Results: Superoxide anion production was enhanced in response to both PMA and fMLF. For PMA there was a peak response at 12-24 hours after IFN-γ then a reduction back to baseline at the lowest dose. For the other three doses there appeared a second increase, not as great as the first, which peaked at 36-48 hours and returned to baseline by 96 hrs. The fMLF response was similar to PMA, but the early peak occurred at 8 hours and returned to baseline at the lower two doses. At 50 and 100 µ/m2 doses a second peak was seen at 24-48 hours with return to baseline by 96 hours. Dose response increases in both peaks of activity were noted for the first two doses and then reached a plateau for the higher doses of both stimuli. Plasma levels of IL-10 peaked at 4-8 hours returning to baseline by 12 hours for the first two doses of IFN-γ and 24 hours at the highest two doses. There was a clear cut dose response effect for peak levels achieved ranging from 60-80 pg./ml at the lowest dose of IFN-γ to 200-600 pg./ml at the highest. Neopterin levels peaked by 24 hours with all doses of IFN and continued to remain elevated to 72 hours moving back to baseline by 96 hours. Evaluation of gene expression is ongoing with analysis by patient, time and dose of IFN-γ with the goal of summarizing the results for the group as a whole. We will evaluate genes which show a 2-fold or greater increase and focus on those genes showing significant expression in a previously analyzed in vitro system of IFN-γ effect during neutrophil maturation of a myeloid cell line with DMSO. These include genes known to be involved in classical aspects of neutrophil function, i.e. transmigration, chemotaxis, phagocytosis and pathogen killing; genes involved in neutrophil clearance and homeostasis, including apoptosis; genes encoding innate immune receptors; and genes encoding guanylate binding proteins, a family of GTPases implicated in antimicrobial activity in different cell types. Conclusion: INF-γ has dramatic in-vivo effects on neutrophils. Dose and time related activities vary and results will help better define the optimal use of the drug. Understanding the nature of INF-γ related transcription activity will help define its clinical effects in CGD and extend the possible uses of this drug to other diseases. Disclosures No relevant conflicts of interest to declare.

Genetic modification of hemopoietic progenitor cells ex vivo, followed by the infusion of the genetically modified cells into the human immunodeficiency virus-1 (HIV-1) infected donor, has been proposed as a treatment for HIV-1 infection. The current study was undertaken to evaluate the effect of hemopoietic stem cell mobilization and harvesting on HIV-1 replication in persons with HIV-1 infection. Eighteen HIV-1–infected persons received recombinant granulocyte colony-stimulating factor (G-CSF; Filgrastim) 10 μg/kg per day, for 7 days. On days 4 and 5, peripheral blood mononuclear cells were harvested by leukapheresis. The CD4+ lymphocyte count at entry was >500/μL for 6 subjects, 200 to 500/μL for 6 subjects, and 0.6 log10) above baseline between days 4 and 7 and returned to baseline by day 27. Significant increases of plasma HIV-1 RNA levels occurred in 5 subjects despite 3-drug antiretroviral therapy. Changes in CD4+ and CD34+ cells during mobilization and harvesting were similar in all subjects whether they had or did not have increased plasma HIV-1 RNA levels. Thus, mobilization and harvesting of bone marrow progenitor cells from persons infected with HIV-1 induced a transient increase in viral replication in some patients but was not associated with adverse effects. (Blood. 2000;95: 48-55)

Granulocyte colony-stimulating factor (r-met Hu G-CSF; filgrastim; 10 microgram/kg/day for 7 days) was used to mobilize CD34+stem cells into the peripheral blood of human immunodeficiency virus type 1 (HIV-1)-infected individuals and a group of HIV-1-uninfected donors as a measure of immunologic reserve in HIV-1-infected people. G-CSF mobilized CD34+ cells of HIV-1-infected individuals with cell counts500 CD4+ cells/mm3, as well as in HIV-1-uninfected donors. In contrast, CD34 cell mobilization was significantly blunted in HIV-1-infected individuals with cell counts500 CD4+ cells/mm3 (200 cell days vs.650 cell days, P.0005, compared with the500 CD4+ cell cohort). At least 1.75x10(7) CD34 cells were harvested by leukapheresis from patients in each study cohort. CD34+ cell viability and the ability to differentiate precursor cells into myeloid and erythroid progenitor cells were not affected by HIV-1 infection.

We have studied the effects of granulocyte colony-stimulating factor (G-CSF) administration to normal individuals on a variety of functional and biochemical neutrophil characteristics that relate to host defense. G-CSF adversely affected neutrophil (polymorphonuclear leukocyte [PMN]) chemotaxis. While this could be partially explained by reduced assembly of neutrophil F-actin, we also recognized an elevated cytosolic calcium mobilization and a normal upregulation of neutrophil CD11b. G-CSF resulted in reduced PMN killing of Staphylococcus aureus with a 10:1 (bacteria:neutrophil) ratio and normal killing with a 1:1 ratio. In association with this, we demonstrated divergent effects on the respiratory burst of intact cells and divergent effects on the content of marker proteins for neutrophil granules. While G-CSF may have resulted in increased content of cytochrome b558 in the cell membrane, it did not alter the amounts of cytosolic oxidase components. After therapy, there was normal content of the azurophilic granule marker, myeloperoxidase, decreased content of the specific granule marker, lactoferrin, and normal content of lysozyme (found in both granules classes). Finally, G-CSF therapy markedly reduced the apoptotic rate of the isolated neutrophil. Therefore, considering disparate functional and biochemical activities, the real benefit of G-CSF therapy may lie in enhanced number and survival of neutrophils.

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.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by absence or dysfunction of the platelet integrin alpha(IIb)beta3. Patient RM is a thrombasthenic variant whose platelets fail to aggregate in response to physiological agonists, despite the fact that they express abundant levels of alpha(IIb)beta3 on their surface. Binding of soluble fibrinogen or fibrinogen mimetic antibodies to RM platelets did not occur, except in the presence of ligand-induced binding site (LIBS) antibodies that transformed the RM integrin complex into an active conformation from outside the cell. Sequence analysis of PCR-amplified genomic DNA and platelet mRNA revealed a C2268T nucleotide substitution in the gene encoding the integrin beta3 subunit that resulted in an Arg724Ter mutation, producing a truncated protein containing only the first eight of the 47 amino acids normally present in the cytoplasmic domain. Functional analysis of both RM platelets and CHO cells stably expressing this truncated integrin revealed that the alpha(IIb)beta3Arg724Ter complex is able to mediate binding to immobilized fibrinogen, though downstream events, including cytoskeletally-mediated cell spreading and tyrosine phosphorylation of focal adhesion kinase, pp125FAK, fail to occur. These studies establish the importance of the membrane-distal portion of the integrin beta3 cytoplasmic domain in bidirectional transmembrane signaling in human platelets, and the role of integrin signaling in maintaining normal hemostasis in vivo.

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.

Infection is a major cause of morbidity and mortality in patients after thermal injury. This predisposition to infections is related, in part, to abnormal polymorphonuclear leukocyte (PMN) function and a diminished respiratory burst. To evaluate the biochemical basis for the defective respiratory burst after major burns, the status of the oxidase enzyme system and its components was investigated. PMNs were isolated from 24 patients with 12% to 62% burns. Oxidase activity of intact PMNs, measured as superoxide anion (O2-) generation or oxygen consumption, was decreased in burn compared with healthy controls. Subcellular fractions from patient PMNs generated less O2- in the sodium dodecyl sulfate cell-free system, and this was related to a diminished contribution by cytosol but not by plasma membrane. Subsequently, cytosol was separated with CM-Sepharose, yielding two fractions; one contained the p47-phox and p67-phox (47/67 mix) and the other contained the remaining cytosolic components (run through [RT]). Although the contribution to oxidase activity made by RT from patient cytosol was similar to that of control, the activity of p47/67 mix from PMNs of burn patients was deficient. Quantitative assays using an immunoautoradiographic technique showed a consistent, but significant decrease in both p47-phox and p67-phox. The addition of purified or human recombinant p47-phox but not p67-phox corrected the diminished oxidase activity of cytosol from burn patients. Thus, decreased respiratory burst activity found in PMNs from individuals with thermal injury was associated with a specific, quantitative deficiency of p47- phox.

Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2−). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2− production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.

Pediatric transfusion medicine (PTM) is a subspecialty of transfusion medicine with no formal training program and few specialists. The Pediatric Transfusion Medicine Academic Awardees (PedsTMAA) group surveyed PTM content experts to identify relevant objectives for the first formal PTM curriculum.Eight North American PTM experts were invited to participate in a two-step consensus process. PTM-related objectives compiled from a review of existing training documents were organized into a survey. Experts were asked to rate each objective for relevancy for a clinical pathology trainee. Content validity indexes (CVIs) and asymmetric confidence intervals (ACIs) of expert ratings and analysis of respondents' comments were used to identify relevant objectives.Six experts participated and reviewed 117 objectives. Based on content validity criteria (CVIor = 0.83 and lower-limit 95% ACIor = 3), a total of 65 objectives were considered relevant. Twenty-three objectives were rated "very relevant" by all the experts while some proposed objectives were determined to be not relevant, out of date, or inappropriate for a resident trainee level.The PedsTMAA group identified 65 objectives for a PTM curriculum. Twenty-three represent a clear core set of objectives and should be considered for clinical pathology training. The next step is to consider the teaching strategies and evaluation methods that will be employed to best deliver this content addressing competency in medical knowledge.

Phthalate esters (PE's) are plasticizers used to soften PVC-based medical devices. PE's are the most abundant man-made pollutants and increase the risk of developing an allergic respiratory disease or a malignancy. The leaching of PE's in donated packed red blood cells (PRBC) during storage was assessed. PRBC transfusion bags containing CPD/AS-1 (ADSOL) buffer were analyzed. Samples were collected on storage day 1 and day 42. Two PE's, di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP), were measured by liquid chromatography coupled to mass spectrometry (LCMS). Interleukin-8 (IL-8) was measured by standard ELISA techniques. DEHP significantly increased from 34.3 microM (+/-20.0 SD) on day 1 to 433.2 microM (+/-131.2 SD) on day 42, a 12.6-fold increase. Similarly, MEHP significantly increased from 3.7 microM (+/-2.8 SD) on day 1 to 74.0 microM (+/-19.1 SD) on day 42, a 20.2-fold increase. Also, DEHP and MEHP increased the release of IL-8 from human umbilical vein endothelial cells (HUVEC). The transfusion of older units of PRBC could lead to an accumulation of PE's possibly resulting in inflammation and other effects. This accumulation could be exacerbated due to the decreased metabolism of PE's since trauma patients have a lower esterase activity, the enzymes responsible for metabolizing PE's. The effect of oxidative stress caused by PE's is discussed as a potential mechanism for increases in inflammation caused by older units of PRBC.

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.

Background The cytokine and drug interferon-γ enhances superoxide anion production by the antimicrobicidal Nox2 enzyme of neutrophils. Because mature neutrophils have a short lifespan, we hypothesized that the effects of interferon-γ on these cells might be mediated by its prolonged exposure to differentiating neutrophil precursors in the bone marrow rather than its brief exposure to mature circulating neutrophils. Effects of INF-γ on Nox2 Activity To address this possibility we exposed the myeloid PLB-985 cell line to interferon-γ for 3 days in the presence of dimethyl sulfoxide which induces terminal differentiation of these cells. Interferon-γ was found to enhance superoxide production by Nox2 in a concentration dependent manner. In contrast, application of interferon-γ alone for 3 days failed to induce detectible Nox2 activity. Additionally, application of interferon-γ for 3 hours to pre-differentiated PLB-985 cells, which models studies using isolated neutrophils, was much less effective at enhancing superoxide anion production. Effects of INF-γ on phox Protein Levels Addition of interferon-γ during differentiation was found to upregulate the Nox2 proteins gp91phox and p47phox in concert with elevated transcription of their genes. The p22phox protein was upregulated in the absence of increased transcription presumably reflecting stabilization resulting from binding to the elevated gp91phox. Thus, increased levels of gp91phox, p47phox and p22phox likely account for the interferon-γ mediated enhancement of dimethyl sulfoxide-induced Nox2 activity. In contrast, although interferon-γ alone also increased various phox proteins and their mRNAs, the pattern was very different to that seen with interferon-γ plus dimethyl sulfoxide. In particular, p47phox was not induced thus explaining the inability of interferon -γ alone to enhance Nox2 activity. Short application of interferon-γ to already differentiated cells failed to increase any phox proteins. Conclusions Our findings indicate that interferon-γ has complex effects on phox protein expression and that these are different in cells undergoing terminal differentiation. Understanding these changes may indicate additional therapeutic uses for this cytokine in human disorders.

Introduction: Prdx6, a member of a new family of peroxidases, is found in neutrophil cytosol associated with p67phox. This protein reduces H2O2 with a conserved cysteine (C47) and also expresses PLA2 activity. In human neutrophils, Prdx6 translocates to plasma membrane after activation and enhances Nox2 activity in a cell-free system of oxidase activity. In transgenic K562 cells and PLB-985 cells, suppression of Prdx6 by siRNA or shRNA decreases Nox2 activity. In these studies, we investigated the association of Prdx6with autophagy in PLB-985 and HEK-293 cells. Methods: PLB-985 and HEK-293 cells were cultured under standard conditions and were transfected with a plasmid producing stable expression of an shRNA targeting Prdx6. PLB-985 cells were matured with the addition of 1.3% DM50 for 4 days. DNA for wild type (WT) Prdx6 and mutants for the Prdx6 active site (C47S) and PLA2active sites (H26A, S32A, S140A) were cloned into pcDNA3.1 which also had silent mutations making all corresponding mRNAs resistant to shRNA targeting. Prdx6 knockdown and vector control cells had stable re-expression of the various proteins when cultured with appropriate antibiotics. Superoxide anion (O2-) was determined by SOD inhibitable chemilluminescence with Digenes (National Diagnosistics). Proteins from lysates of various cell lines were separated by 10-12% SDS-PAGE and blotted onto nitrocellulose. Specific proteins were detected standard antibodies and a chemilluminescent technique with quantitation completed using Image J platform. Autophagy in PLB-985 cells with or without suppression of Prdx6 was completed by expression of LC3-II by Western blot after stimulation with serum opsonized zymsoan (5mg/mL). In HEK-293 cells, autophagy was determined by quantification of LC3-II (western blot) under based conditions with the addition of 10µM Chloraquine or starvation conditions with minimal media. Results: Stable suppression of Prdx6 by shRNA was achieved in both PLB-985 cells (down to 30%) and HEK cells (25%). Re-expression of WT and Prdx6 mutant proteins re-established normal levels. O2-production in response to lµM FMLP was decreased by 44% in KD PLB-985 cells compared to control and re-establishment of oxidase activity depended on PLA2, not Prdx activity. In PLB-985 cells, LC3-II levels increased (seven fold) over the first 30 minutes after exposure to opsonized zymosan, decreasing to baseline at 60 minutes. Prdx6 suppressed PLB-985 cells showed no change in LC3-II after opsonized zymosan. In HEK-293 cells lacking an active oxidase, suppression of Prdx6 resulted in increased autophagy under basal and starvation conditions. Furthermore, in HEK-293 cells, reintroduction of empty vector or Prdx6 mutated at the Prdx active site (C47S) did not suppress autophagy under basal conditions. However, re-introduction of WT and mutant Prdx6 at the PLA2 active site (D140A) did suppress autophagy demonstrating that this effect was associated with Prdx activity. Similar results were obtained under starvation conditions. Conclusions: In neutrophil like cells Prdx6 enhanced Nox2 activity through its PLA2 activity and suppression of Prdx6 decreased autophagy as measured by LC3-II accumulation after exposure to a phagocytic stimulus. In non-myeloid HEK-293 cells, suppression of Prdx6 was associated with increased autophagy under basal and starvation conditions, and this effect was dependent on Prdx activity. Prdx6, then, plays a critical role in neutrophil functions such as Nox2 activity and autophagy. Prdx6 may also be involved in autophagy in non-myeloid cells, and the mechanism for its involvement in autophagy may relate to different activities expressed by this protein. Disclosures No relevant conflicts of interest to declare.

Introduction: Most information about the immunoregulatory functions of INF-γ has focused on the interaction with the adaptive immune system and less is reported about neutrophils. The majority of studies with neutrophils evaluate the effects of INF-γ on differentiated cells. Since an important issue INF-γ treatment is the function of neutrophils developed under the influence of this cytokine, we investigated the effects of INF-γ on a myeloid cell line that can be differentiated into neutrophil-like cells. Methods: PLB-985 cells were grown under standard culture conditions (5% CO2, 37oC) with RPMI and 10% bovine serum and were matured with the addition of 1.3% DMSO in the presence and absence of various concentrations of INF-γ-1b (Vidara Therapeutics Ltd.). After 72 and 96 hours, the cells exhibited neutrophil-like phenotype. As controls, cells were incubated with media or media plus INF-γ alone. Superoxide anion (O2-) production was measured as S00 inhibitable cytochrome c reduction after exposure to PMA (200 ng/mL) or FMLP (1 µM). Cell lysates were prepared in the presence of 0.1% SDS, 1% Triton X 100 and a protease inhibitor mix. Proteins from the lysate were separated on 10-15% SDS PAGE, blotted onto PVDF or nitrocellulose, exposed to primary antibodies for phox proteins overnight at 4oC and then appropriate peroxidase labeled secondary antibodies. Protein/antibody complexes were detected with an ECL detection system and were quantitated by densitometry with Image J software. Results: PLB-985 cells cultured for 4 days with 30ng/ml ( 600 IU/ml) INF-γ, DMSO or DMSO+ INF-γ were stimulated with PMA or fMLF and O2- production was measured over 5 minutes (for fMLF) or 10 minutes (for PMA). INF-γ alone resulted in minimal O2- generation. For fMLF a significant (p When a range of INF-γ concentrations (5-60 ng/ml) were co-applied during DMSO mediated PLB-985 differentiation over 3 days, the increase in O2-production was concentration dependent and reached a plateau at 30ng/ml for both stimuli. Culture of cells with INF-γ alone resulted in dramatic increases in p22phox and gp91phox but failed to cause more than minimal increases in the initially low levels of p40phox, p47phox and p67phox accounting for the inability of INF-γ alone to induce oxidase activity. Differentiation of the cells with DMSO caused a small decrease in p22phox and a small increase in gp91phox but also resulted in large increases in p40phox, p47phox and p67phox which would explain the ability of DMSO to induce Nox2 activity. Inclusion of INF-γ with DMSO further increased p47phox, and increased p22phox and gp91phox relative to DMSO alone, potentially explaining the enhanced Nox2 activity noted above. In striking contrast, when INF-γ was applied to cells first differentiated with DMSO, a minimal increase in fMLF induced O2- production was seen and only a small increase in PMA induced O2-generation was determined. Consistent with this, INF-γ applied to pre-differentiated PLB-985 cells did not enhance p40phox, and gp91phox, minimally increased p22phox, and decreased p47phox and p67phox slightly. Conclusions: These studies demonstrate that INF-γ induces very different phenotypes in myeloid cells depending on the differentiation level of the cell. Dramatic differences were noted whether the INF-γ was present during maturation of these myeloid cells as opposed to after differentiation and include major differences in expression of phox proteins as well as activity of the Nox2 enzyme system. These data not only support the administration of INF-γ for specific neutrophil dysfunction disorders, but also suggest, in theory, expanded uses of this cytokine. Disclosures No relevant conflicts of interest to declare.

CP-64131 (CP), an aminobenzazepine with cytokine-like, physiologic effects similar to granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage (GM)-CSF, increases the number of neutrophils and stimulates marrow recovery after doxirubicin ablation. CP can also function as a neutrophil agonist, like formyl-Met-leu-Phe (fMLP). In these studies, we show that CP is unique in that it stimulates the p38-mitogen-activated protein kinase (MAPK) pathway but not extracellular signal-regulated kinase (ERK)1/2 or c-jun N-terminal kinase MAPKs in human neutrophils from peripheral blood. This is in contrast to other neutrophil agonists such as fMLP, interleukin (IL)-8, or GM-CSF, which stimulate multiple MAPK pathways. Like fMLP and IL-8, CP is capable of stimulating superoxide (O2−) production, CD11b expression, and cell polarization in human neutrophils. CP-stimulated O2− production is completely dependent on p38-MAPK activation, as determined by sensitivity to the p38-MAPK inhibitor SB203580. In contrast, SB203580 only partially inhibits expression of CD11b and has no effect on cell polarization stimulated by CP. Therefore, CP treatment of neutrophils activates p38-MAPK but has effects independent of p38-MAPK activation. In human embryonic kidney 293 cells, a human kidney epithelial cell line CP stimulates p38-MAPK and modestly activates ERK1/2. The findings define CP as a novel, small molecule, which has little cellular toxicity in vitro. CP has the ability to activate specific MAPK pathways in different cell types and should prove to be an effective agonist in combination with inhibitors to study biological responses regulated by MAPKs.

A mixture of lysophosphatidylcholines (lyso-PCs) are generated during blood storage and are etiologic in models of acute lung injury. We hypothesize that lyso-PCs stimulate polymorphonuclear neutrophils (PMNs) through Ca2+-dependent signaling. The lyso-PC mix (0.45–14.5 μM) and the individual lyso-PCs primed formyl-Met-Leu-Phe (fMLP) activation of the oxidase (1.8- to 15.7-fold and 1.7- to 14.8-fold; P

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of hemotherapy. We report a series of 90 TRALI reactions in 81 patients secondary to transfusion with whole blood platelets (72 reactions), apheresis platelets (2), packed red cells (15), and plasma (1). The overall prevalence was 1 in 1120 cellular components. To examine the epidemiology of TRALI, we completed a nested case-control study of the first 46 patients with TRALI compared with 225 controls who had received transfusions. We then completed a prospective analysis of possible biologic response modifiers responsible for 51 of the TRALI cases, including human leukocyte antigen (HLA) class I, class II, and granulocyte antibodies in donors and neutrophil (PMN) priming activity in the plasma of the implicated units and recipients. Two groups were at risk: patients with hematologic malignancies (P

This study examined the effectiveness of 3 leukocyte-reduction (LR) methods in depleting the residual level of cytomegalovirus (CMV) in blood products measured by quantitative polymerase chain reaction (QA-PCR). At 2 locations over 3 allergy seasons, apheresis platelets and whole blood were collected from 52 healthy CMV seropositive subjects having an elevated titer of CMV DNA (median = 2400 genome equivalents [GE]/mL) resulting in 32 evaluable LR apheresis platelets, 31 filtered platelets from whole blood, and 31 filtered red blood cells (RBCs) from whole blood. Leukoreduction by apheresis and filtration resulted in substantial reduction of detectable CMV DNA levels with 99.9% of the LR products expected to have less than 500 GE/mL of CMV DNA. No difference was found between methods (P = .52). CMV genomic leukocyte subset localization was determined by QA-PCR of fluorescence-activated cell sorter (FACS)-sorted peripheral blood from 20 seropositive subjects (n = 10 > 100 GE/mL, n = 10 QA-PCR negative). CMV was detected in monocyte (13 of 20) and granulocyte (3 of 20) fractions. Presence of competent virus in QA-PCR positive (> 100 GE/mL) peripheral blood samples was verified with 4 of 19 subjects positive in shell vial assay, and 8 of 18 positive for CMV gene products (messenger RNA). We observed a seasonal DNAemia variation in seropositive subjects. CMV seropositive subjects (n = 45) entered into longitudinal monitoring in March/April 1999 were QA-PCR negative at baseline. Subjects converted to a positive QA-PCR coincident with increased seasonal allergen levels (Norfolk 15 of 18 evaluable in 43.4 ± 9.48 days; Denver, 16 of 23 evaluable in 96 ± 26.3 days). These data demonstrate effective reduction of CMV load by LR during periods of DNAemia in CMV seropositive subjects.

A 5-week-old male infant presented with severe bacterial infections and poor wound healing, suggesting a neutrophil defect. Neutrophils from this patient exhibited decreased chemotaxis, polarization, azurophilic granule secretion, and superoxide anion (O2−) production but had normal expression and up-regulation of CD11b. Rac2, which constitutes >96% of the Rac in neutrophils, is a member of the Rho family of GTPases that regulates the actin cytoskeleton and O2−production. Western blot analysis of lysates from patient neutrophils demonstrated decreased levels of Rac2 protein. Addition of recombinant Rac to extracts of the patient neutrophils reconstituted O2−production in anin vitroassay system. Molecular analysis identified a point mutation in one allele of the Rac2 gene resulting in the substitution of Asp57 by an Asn (Rac2D57N). Asp57 is invariant in all defined GTP-binding proteins. Rac2D57Nbinds GDP but not GTP and inhibits oxidase activation and O2−productionin vitro. These data represent the description of an inhibitory mutation in a member of the Rho family of GTPases associated with a human immunodeficiency syndrome.

Abstract 1179 Background Previous studies by our laboratory have demonstrated changes in apheresis collected, PLT concentrates during storage that are enhanced by Mirasol®PRT treatment. These include increased expression of P-selectin on PLTs and increased formation of neutrophil (PMN)/PLT aggregates. To explore the effect of these changes in a microvascular environment, we measured PLT adherence in a microfluidic chamber coated with collagen. Methods Three, double apheresis platelet products were collected from healthy volunteers with TRIMA system under an IRB approved protocol. One product of each pair was treated with Mirasol PRT and both were stored under standard conditions. Aliquots were sterilely removed from products during storage. To 1000 μl of fresh, ABO type specific, citrate anticoagulated whole blood was added 60 μl of apheresis platelet concentrate with or without PRT treatment on day 1 or 7 of storage and incubated for 5 min at 37C. The sample was recalcified with 20 mM CaCl2 and then pulled through the inlet of a microfluidic chamber at a flow sheer rate of 100 s−1. The flow chamber was composed of two parts, a glass slide patterned with collagen and a series of four polydimethylsiloxane microfluidic chambers per inlet. Stored PLTs were labeled with fluorescently labeled anti-CD41 to distinguish from PLTs of the whole blood. The chamber was mounted on an inverted fluorescence microscope, and video microscopic images sampled on observation field every 7 sec over 7 min. The number of fluorescent cells (stored PLTs) per field and the total area covered by all PLTs were determined at 7 min. Results The area, expressed as % of total field, covered by all of the PLTs in the sample was not different for whole blood alone (29 ± 3); Day 1 untreated (23 ± 9); Day 1 PRT treated (26 ± 8); Day 7 untreated (21 ± 5); and Day 7 treated (25 ± 9) PLTs. In contrast, there was an increase in the number of treated apheresis PLTs at Day 1 compared to untreated apheresis platelets binding to the chamber (2.26 ± 0.61 untreated, vs. 5.45 ± 1.45 treated, numbers are mean ± SEM for numbers of cells adhered normalized to the PLT count in the product, significant, p=0.0229). There was also an increase in binding of treated compared to untreated PLTs at Day 7 (2.43 ± 0.47 untreated, vs. 4.42 ± 1.35 treated). However, the results for untreated PLTs on Day 1 and 7 and treated PLTs on study days were not different. The increased binding of Mirasol PRT treated PLTs parallels the small decrease in recovery seen in clinical studies of patients receiving treated PLT concentrates compared to untreated products. Conclusion PRT treatment increases adherence of PLTs early in storage but does not appear to progress further. The changes may have implications for clinical responses to platelet transfusions. Evaluation of adherence in the microfluidic chamber provide a model which simulate the microvascular environment. Disclosures: Ambruso: Terumo BCT: Research Funding. Seewald:Terumo BCT: Research Funding. Marschner:Terumo BCT: Employment. Goodrich:Terumo BCT: Employment.

Abstract 2288 Introduction Platelet concentrates develop biologically active compounds during storage which may play a role in adverse events of transfusion. Although many studies have focused on release of soluble pro-inflammatory compounds, changes in cells such as platelets may contribute to the response or pro-inflammatory effects of transfusion products. In previous studies we have shown that pathogen reduction with Mirasol PRT of apheresis PLT concentrates induced changes in PLTs which increased during storage including expression of P-selectin and the capacity to form PLT-PMN aggregates. These changes were also associated with increased adherence of stored PLTs in a microfluidic chamber. In the current study we evaluated P-selectin and PLT-PMN aggregate formation in whole blood after treatment with Mirasol PRT. Methods Ten units of whole blood from healthy donors were drawn into CPD under an IRB approved protocol. Five units where treated with Mirasol PRT and five units remained untreated. Aliquots were taken before and after treatment (or at same times for untreated units) on Day 0 and 24–27 hours after draw on Day 1 after storage at 22–24°C. Platelet rich plasma (PRP) was produced by standard technique and PLTs isolated on a Sepharose 2B column. PLTs were incubated with APC-CD62P and FITC-CD41a for comparison with P-selectin expressed as percent positive increase over isotype control. PMNs were isolated from 50 ml of heparinized peripheral blood drawn from healthy, ABO-matched donors. After a 5 min pre-incubation at 37°C in a shaking water bath, untreated, treated PLTs, and PMNs were incubated separately or together for 3 min then quenched at 4°C. FITC-CD41a and FITC-IgG1 isotype for platelet-PMN aggregate quantitation or PE-CD11b for CD11b expression on neutrophils were added. Platelet-PMN aggregates was expressed as percent of all PMN events above CD41a isotype control. CD11b expression was determined for PMNs after exposure to PLTs by flow cytometry. Results P-selectin positive cells did not change significantly over time in samples from untreated products (Table 1). After Mirasol PRT treatment, cells positive for P-selectin increased on Day 0 and 1 (p Results for PMN-PLT aggregates parallel expression of P-selectin (Table 2). No increase in aggregate formation was seen for untreated samples; however, treated samples showed increased aggregates at Day 0 and 1 (p Conclusion Treatment of whole blood with Mirasol PRT results in increased expression of P-selectin on PLTs and capability of forming aggregates with PMNs. The changes occur soon after initiation of treatment, and the implications for clinical response to of platelet infusion and adverse events of transfusion need to be established. Disclosures: Ambruso: Terumo BCT: Research Funding. Seewald:Terumo BCT: Research Funding. Marschner:Terumo BCT: Employment. Goodrich:Terumo BCT: Employment.

Abstract 2139 Introduction The phagocyte NADPH oxidase (phox) is a multiprotein, transmembrane-enzyme-complex found in diverse professional phagocytes including neutrophils. Phox in its inactive state consists of the membrane-associated subunit cytochrome b558 (a dimer of gp91phox/Nox2 and p22phox) and cytoplasmic subunits which include p47phox, p67phox, p40phox and Rac1 or Rac2. Phosphorylation of phox proteins promotes tight interaction of the cytoplasmic phox components with cytochrome b558 and the resulting active complex catalyzes transmembrane electron transfer from cytoplasmic NADPH to molecular oxygen forming superoxide anion (O2−). O2− and other reactive oxygen species formed from it are microbicibal and crucial to pathogen killing by neutrophils. Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with a peroxiredoxin activity that reduces oxidized lipids and H2O2 and a PLA2 activity that cleaves the sn2 position of phospholipids forming the corresponding free fatty acid and lysophospholipid. Previous work in our laboratory has identified Prdx6 as a binding partner of p67phox and as an enhancer of O2− production by active phox; we have shown that this enhancement is due to products of the PLA2 activity of the peroxiredoxin but the precise mechanism by which these molecules act is unclear. Here we present evidence that Prdx6 causes maximal activation of phox when the complex is activated by physiological agonists (the formylated peptide fMLP and the particulate agonist serum opsonized zymosan (SOZ)) but not when it is activated by the non-physiological agonist phorbol 12-myristate 13-acetate (PMA). Because fMLF and SOZ interact with cell surface receptors but PMA enters the cell and activates protein kinase C (PKC) enzymes that directly phosphorylate phox proteins our data provides a clue as to the mode of action of Prdx6 in phox activation. Methods PLB985 cells were used to model neutrophils. This myeloid cell line can be terminally differentiated to a neutrophil like state with phox activity by exposure to DMSO. Prdx6 was suppressed in these cells by stable expression of an shRNA resulting in an approximately 70% reduction of the protein. Following maturation of the cells they were stimulated with fMLP, SOZ or PMA and O2− production was measured by superoxide dismutase (SOD) inhibitable luminescence of Diogenes fluorophore. For fMLP, due to the transient nature of the O2- production, the entire amount of O2− production was measured. SOZ and PMA induced prolonged (>1hr) O2− production and O2− production over 1hr was thus measured. Results In cells with Prdx6 suppressed, SOD inhibitable O2− production in response to fMLP was reduced by 53% ± 3% (SEM), n=34, which was significant (p In contrast, the response to PMA was not altered (p>0.5, n of at least 4) at a range of PMA concentrations from one causing slight activation of phox (10 ng/ml) to one causing maximal O2- production (1 μg/ml). Conclusions fMLP binds to receptors on neutrophils and triggers intracellular signaling cascades that ultimately lead to phosphorylation and activation of phox. Similarly, opsonins of particulate stimuli bind cell surface complement receptors that trigger intracellular signaling cascades that activate phox. PMA on the other hand enters cells and activates PKC enzymes that directly phosphorylate phox proteins; it thus bypasses the physiological signaling pathways that mediate the fMLP and SOZ response. Since Prdx6 enhances the SOZ and fMLF mediated phox response but not the PMA mediated response we propose that in myeloid cells, products of the PLA2 activity of Prdx6 enhance the SOZ and PMA mediated signaling cascades. This may be via activation of signaling molecules in these pathways which would be consistent with the known ability of the fatty acid arachidonate to activate PKC enzymes, MAP kinases, PLA2 enzymes and a class Ia phosphatidylinositol 3-kinase and to mobilize Ca2+. Disclosures: No relevant conflicts of interest to declare.

Immune-mediated thrombocytopenia and neutropenia are not uncommon problems in the newborn period. Such cytopenias have been associated with increased levels of IgG, IgM, or both, in the serum, documented by indirect assay, and/or on the cell surface, documented by direct assay, and decreased cell survival. However, interpretation of measurement of platelet- or neutrophil-associated antibodies is problematic due to the lack of data from healthy neonates. In this study, both direct and indirect neutrophil- and platelet-associated IgG and IgM were measured in cord samples from 44 healthy, term neonates. These infants had increased amounts of direct platelet-associated IgG and direct neutrophil-associated IgM and IgG compared with adult controls. Serum samples from these healthy newborns manifested a significantly decreased level of IgM binding to target platelets compared with serum from healthy adult controls. There was not a significant difference in direct platelet-associated IgM, or indirect Ig to neutrophils or platelets. Complete blood counts drawn at 24 h of age were within normal limits in 34/35 infants studied. Moreover, there was not a statistical difference in platelet or neutrophil Ig studies between the newborns of multiparous and primigravida mothers. The physiologic consequences of the increased amounts of these Ig to the survival and function of platelets and neutrophils in neonates is unclear. However, these values must be considered for proper interpretation of platelet and neutrophil Ig measurements in newborns with cytopenias.

Abstract 1123 Introduction: Platelet concentrates develop biologically active compounds during storage which may play a role in adverse events of transfusion. Although many studies have focused on release of soluble pro-inflammatory compounds, changes in cells such as platelets may contribute to the pro-inflammatory effects of transfusion products. We evaluated untreated and Mirasol Pathogen Reduction Technology (PRT) treated platelet concentrates for expression of P-selectin and interactions between platelets and PMNs. Methods: Four, double apheresis platelet products were collected from healthy volunteers with TRIMA system under an IRB approved protocol. One product of each pair was PRT treated with Mirasol PRT and both were stored under standard conditions. At various times, aliquots were sterilely removed from products. Expression of P-selectin on platelets was defined with a specific antibody. Neutrophils, isolated from ABO type specific peripheral blood of volunteers, were incubated with platelet concentrates for 3 min at 37° C, and labeled CD41 was added. PMNs were identified with flow cytometry by forward/side scatter and platelets by fluorescence and the % of PMNs associated with platelets determined. Inhibition of platelet/neutrophil interaction with antibodies to adhesion molecules was also examined. In parallel experiments, activation of neutrophils after incubation with platelets was determined by oxidation of dihydrorhodamine (DHR). Results: During storage, platelet expression of P-selectin increased in both untreated and Mirasol treated platelet concentrates (p Conclusion: During storage, platelets exhibit increased P-selectin expression and the capability to adhere to PMNs. These effects were greater after Mirasol PRT treatment. The cell association depended on the interaction between P-selectin (platelets) and PSGL-1 (PMNs). These changes occurring during storage of platelet concentrates may explain platelet recovery patterns in patients requiring repeated transfusions with untreated and PRT treated products and could play a potential role in adverse events for either product type. Disclosures: Goodrich: CaridianBCT Biotechnologies: Employment, Patents & Royalties, Research Funding. Marschner:CaridianBCT Biotechnologies: Employment, Research Funding.

Abstract 926 Introduction: An intact respiratory burst is critical to the microbial activity of neutrophils. The respiratory burst is initiated by assembly and activation of the NADPH oxidase, Nox2. We have identified a 29 kDa protein from human neutrophils (p29) which binds to the oxidase component p67phox and enhances superoxide anion (O2-) production in a cell-free, NADPH activity oxidase system. Identified as Prdx6 by sequence and was found to have Prdx activity by the recombinant protein. p29 Prdx6 also has a calcium-independent PLA2 activity that is optimal at low pH (aiPLA2). Although Prdx6 is found in many tissues, its function in myeloid cells is not well established. p29 Prdx6 was knocked down in a myeloid cell line (PLB-985) and the effect on the Nox2 activity of theses cells was investigated. In addition, wild type p29 Prdx6 or mutants with altered Prdx and PLA2 active sites were re-expressed in the knockdown cells, to explore the possibility that at least one of the catalytic activities is involved in enhancing the oxidase. Methods: PLB-985 cells were transfected with a plasmid encoding a p29 Prdx6 targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx6 was confirmed by Western blot, but no change in the other known oxidase components was detected. cDNAs for wild type (wt) p29 Prdx6 and known mutants for the Prdx activity site (C47S) and PLA2 active site (H26A and S32A) were cloned into pcDNA3.1 producing plasmids generating mRNAs resistant to shRNA. Knockdown cells transfected with these plasmids were cultured with both puromycin and G418 with re-expression of wt or mutant Prdx6 to comparable levels in PLB985 cells, documented by Western blot. After maturation of the knockdown, Prdx re-expressing, and control cells with DMSO for 4 days, fMLP (1μM) stimulated O2- production was measured by luminescence of Diogenes probe. Results: Using stable expression of shRNA, p29 Prdx6 protein was reduced to 31±8% (mean ± SEM, n=5) of that in non-knockdown control PLB985 cells. O2- production in response to fMLP was reduced (Student's t-test, p Disclosures: No relevant conflicts of interest to declare.