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Children with Down syndrome (DS) are more likely to have hematologic and immunologic abnormalities compared to their typically developing peers, but normal ranges have not been defined. The goal of this study was to create references for complete blood counts (CBCs) in patients with DS.A retrospective investigation of 355 (male = 196, 55.2%; mean age = 6.49 years, SD = 5.07) healthy pediatric patients with DS who received a CBC between 2011 and 2017 as part of their medical care at a single, large, pediatric teaching hospital. Control data on 770 healthy patients without DS were included. Descriptive statistics were performed on demographic and clinical characteristics. Kruskal-Wallis H tests, nested analysis-of-variance tests, and t-tests were run to determine the significant associations.Age-related normative curves for healthy children with DS outlining 2.5th, 25th, 50th, 75th, and 97.5th percentiles are provided for total white blood count, hemoglobin concentration, hematocrit, mean corpuscular volume, and platelet, absolute neutrophil, absolute lymphocyte, eosinophil, monocyte, and basophil counts. Statistical differences were found between children with and without DS receiving care at the same hospital based on matched age/sex groups.This study demonstrates that patients with DS have different reference ranges for multiple blood counts compared to those without DS, creating a new resource for pediatricians to refer to when evaluating CBCs in this population.

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

Primary immunodeficiency states are not uncommon disorders which can affect the innate or adaptive arms of the immune system. All of these diseases have the potential for adverse pulmonary manifestations, including infections and noninfectious complications. In this chapter, we review the major diseases of the innate and adaptive immune system with a focus on their effects on the lung. Included are descriptions of the disorders, laboratory and diagnostic findings, associated cellular and biochemical effects, genetic abnormalities and inheritance, infectious or noninfectious pulmonary manifestations, and management approaches.

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.

Background Granulocyte transfusion from healthy donors is used in the treatment of patients with granulocyte function defects, or transient neutropenia and severe bacterial or fungal infections resistant to maximal antimicrobial treatment. Study Design and Methods This study evaluated the performance and safety of the newly developed granulocyte collection protocol of the Spectra Optia in a prospective, multicenter, open-label, randomized, paired crossover trial compared with the COBE Spectra apheresis system in a population of 32 evaluable healthy subjects. All subjects received granulocyte–colony-stimulating factor and dexamethasone before collection. Results Granulocyte procedures from Spectra Optia apheresis procedures had an approximately 23% higher polymorphonuclear (PMN) collection efficiency (CE) than the COBE Spectra collections (mean, 53.7% vs. 43.2%; p

Hydroxyethyl starch (hetastarch) is a synthetic glucose compound with extensive clinical use as a volume expander. Because of its red blood cell-sedimenting properties, hetastarch plays a major role in preparation of granulocyte products. Recent concerns have been raised about the use of hetastarch in critically ill patients for the development of renal injury and other severe adverse events. In contrast, granulocyte donors receive much less of this compound during collection procedures, and over many years, minimal toxicity has been documented in these individuals. Furthermore, granulocyte products contain very little hetastarch, and ill effects on renal function have not been associated with their administration. This review assesses available information about the toxicity profile of hetastarch in critically ill patients requiring a volume expander as well as granulocyte donors and recipients. Because of the lack of toxicity in these latter two groups, hetastarch should be available for preparation of granulocyte products and their administration.

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.

A male with sickle SC disease presented at age 8 years with proliferative sickle cell retinopathy (PSCR) and bilateral vitreous hemorrhage which spontaneously resolved, then recurred at 13 years of age. Despite conventional therapy with repeated pan-retinal photocoagulation and pars plana vitrectomy, he developed progressive PSCR and recurrent vitreous hemorrhage over the next 30 months. We describe the successful use of chronic red cell exchange transfusion (RCE) to preserve his vision and stabilize the retinopathy.

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.

H. W. Reesink, J. Lee, A. Keller, P. Dennington, J. Pink, R. Holdsworth, H. Schennach, M. Goldman, T. Petraszko, J. Sun, Y. Meng, K. Qian, V. Rehacek, P. Turek, T. Krusius, E. Juvonen, P. Tiberghien, D. Legrand, G. Semana, J. Y. Muller, J. Bux, A. Reil, C. K. Lin, H. Daly, E. McSweeney, L. Porretti, N. Greppi, P. Rebulla, H. Okazaki, S. A. Sanchez-Guerrero, H. A. Baptista-Gonzalez, C. Martinez-Murillo, A. Guerra-Marquez, H. Rodriguez-Moyado, R. A. Middelburg, J. C. Wiersum-Osselton, A. Brand, C. van Tilburg, D. Dinesh, J. Dagger, P. Dunn, E. Brojer, M. Letowska, K. Maslanka, E. Lachert, M. Uhrynowska, E. Zhiburt, M. Palfi, G. Berlin, B. M. Frey, L. Puig Rovira, E. Muniz-Diaz, E. Castro, C. Chapman, A. Green, E. Massey, N. Win, L. Williamson, C. C. Silliman, D. J. Chaffin, D. R. Ambruso, N. Blumberg, P. Tomasulo, K. J. Land, P. J. Norris, O. C. Illoh, R. J. Davey, R. J. Benjamin, A. F. Eder, L. McLaughlin, S. Kleinman & S. Panzer

BACKGROUND: Alloimmunization to red blood cell (RBC) blood group antigens is a major complication for patients with sickle cell disease (SCD), which limits the usefulness of RBC transfusion. Here, we report our experiences with extended RBC antigen matching for SCD patients. STUDY DESIGN AND METHODS: Records for 99 SCD patients transfused only with the extended matching protocol between 1993 and 2006 were reviewed. Patients and donors were phenotyped for 20 blood group antigens and RBC units that were negative for antigens not expressed by the recipient were provided. When necessary, mismatches were allowed at Lea, Leb, Fyb, and MNSs to meet requirements for antigens regarded as the most clinically significant. Matched RBC units (6946) were provided to 99 patients (mean, 70 units/patient; range, 1-519 units/patient). Eliminating mismatches, 90% of the transfusions matched all other negative antigens. RESULTS: Seven alloantibodies were detected in seven patients resulting in 7% alloimmunized at a rate of 0.1 antibodies per 100 units transfused. Three recipients who developed antibodies were D mosaic and would have been mistyped with serologic techniques. Alloimmunization was decreased compared to ABO and/or D matching at our institution and others. Twelve autoantibodies and no severe hemolytic transfusion reactions were reported. CONCLUSION: Exact matching for ABO, Rhesus, Kell, Kidd, and Fya and extending this match whenever possible is an effective strategy to reduce alloimmunization to RBC antigens. Consideration should be given to exploring this conclusion further with a controlled, multi-institutional trial to determine efficacy, cost-benefit analysis, and reproducibility of this approach.

BACKGROUND: Pathogen reduction of platelets (PRT-PLTs) using riboflavin and ultraviolet light treatment has undergone Phase 1 and 2 studies examining efficacy and safety. This randomized controlled clinical trial (RCT) assessed the efficacy and safety of PRT-PLTs using the 1-hour corrected count increment (CCI1hour) as the primary outcome. STUDY DESIGN AND METHODS: A noninferiority RCT was performed where patients with chemotherapy-induced thrombocytopenia (six centers) were randomly allocated to receive PRT-PLTs (Mirasol PRT, CaridianBCT Biotechnologies) or reference platelet (PLT) products. The treatment period was 28 days followed by a 28-day follow-up (safety) period. The primary outcome was the CCI1hour determined using up to the first eight on-protocol PLT transfusions given during the treatment period. RESULTS: A total of 118 patients were randomly assigned (60 to PRT-PLTs; 58 to reference). Four patients per group did not require PLT transfusions leaving 110 patients in the analysis (56 PRT-PLTs; 54 reference). A total of 541 on-protocol PLT transfusions were given (303 PRT-PLTs; 238 reference). The least square mean CCI was 11,725 (standard error [SE], 1.140) for PRT-PLTs and 16,939 (SE, 1.149) for the reference group (difference, −5214; 95% confidence interval, −7542 to −2887; p

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.

BACKGROUND: Pathogen inactivation technologies provide a potential solution to donor screening and blood testing strategies reducing the risk of transfusion-transmitted infectious diseases. The Mirasol pathogen reduction technology (PRT) system (CaridianBCT) uses riboflavin and UV light to introduce modifications in nucleic acids, reducing the infectious pathogen load in blood components. This study evaluated serum of patients who received PRT-treated platelet (PLT) concentrates over a time period of 28 days for the appearance of antibodies to neoantigens on PLTs. STUDY DESIGN AND METHODS: Serum specimens were obtained at study inclusion and at the 28-day follow-up visit from patients randomly assigned to receive PRT-treated PLT concentrates and at study inclusion of control subjects receiving untreated PLTs. PLT samples from untreated and PRT-treated PLTs were collected. PLT samples for each patient were pooled for the analysis. The presence of antibodies in patient serum to neoantigens was determined with a modified Capture-P assay. The presence of auto- or alloantibodies in specific patient samples was determined with PAKAUTO and PAK 12 techniques. RESULTS: Forty-four patients receiving PRT-treated PLTs were evaluated; none of these patients demonstrated antibodies to neoantigens. One patient demonstrated a PLT alloantibody to human PLT antigen (HPA)-5b and autoreactivity to glycoprotein Ia/IIa, consistent with an alloantibody at the beginning, but not at the end of the study interval. Of 22 patients evaluated in the control group, one alloantibody with HPA-5b reactivity was detected. CONCLUSION: Patients receiving PRT-treated PLT concentrates did not demonstrate antibodies to neoantigens, suggesting that neoantigen formation is not a critical side effect of this pathogen reduction process.

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 now the leading cause of transfusion-associated mortality, even though it is probably still underdiagnosed and underreported.The National Heart, Lung, and Blood Institute convened a working group to identify areas of research needed in TRALI. The working group identified the immediate need for a common definition and thus developed the clinical definition in this report.The major concept is that TRALI is defined as new acute lung injury occurring during or within 6 hrs after a transfusion, with a clear temporal relationship to the transfusion. Also, another important concept is that acute lung injury temporally associated with multiple transfusions can be TRALI, because each unit of blood or blood component can carry one or more of the possible causative agents: antileukocyte antibody, biologically active substances, and other yet unidentified agents.Using the definition in this report, clinicians can diagnose and report TRALI cases to the blood bank; importantly, researchers can use this definition to determine incidence, pathophysiology, and strategies to prevent this leading cause of transfusion-associated mortality.

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.

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.

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.

Chemoattractant priming and activation of PMNs results in changes in cytosolic Ca 2+ concentration, tyrosine kinase activity, and gene expression. We hypothesize that the initial signaling for the activation of a 105 kDa protein (Rel-1) requires Ca 2+ -dependent tyrosine phosphorylation. A rapid and time-dependent tyrosine phosphorylation of Rel-1 occurred following formyl-Met-Leu-Phe (fMLP) stimulation of human PMNs at concentrations that primed or activated the NADPH oxidase (10 −9 to 10 −6 M), becoming maximal after 30 s. Pretreatment with pertussis toxin (Ptx) or tyrosine kinase inhibitors abrogated this phosphorylation and inhibited fMLP activation of the oxidase. The fMLP concentrations employed also caused a rapid increase in cytosolic Ca 2+ but chelation negated the effects, including the cytosolic Ca 2+ flux, oxidase activation, and the tyrosine phosphorylation of Rel-1. Conversely, chelation of extracellular Ca 2+ decreased the fMLP-mediated Ca 2+ flux, had no affect on the oxidase, and augmented tyrosine phosphorylation of Rel-1. Phosphorylation of Rel-1 was inhibited when PMNs were preincubated with a p38 MAP kinase (MAPK) inhibitor (SB203580). In addition, fMLP elicited rapid activation of p38 MAPK which was abrogated by chelation of cytosolic Ca 2+ . Thus, fMLP concentrations that prime or activate the oxidase cause a rapid Ca 2+ -dependent tyrosine phosphorylation of Rel-1 involving p38 MAPK activation.

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).

BACKGROUND: Umbilical cord blood is an alternative peripheral blood progenitor cell source for patients who need transplantation. A presumed advantage of cord blood is the ability to increase minority recruitment. STUDY DESIGN AND METHODS: The racial composition of five member cord blood banks of the National Marrow Donor Program (NMDP) was compared, representing 9020 cord blood donors with NMDP marrow donors from comparable geographic areas, representing 417,676 donors. Cord blood and marrow donors self-reported racial designations on questionnaires. Donor statistics were compared with baseline racial data of deliveries from participating hospitals for cord blood donors and with geographic census data for marrow donors. RESULTS: The California, Florida, and Massachusetts cord blood banks recruited a lower percentage of minorities than the corresponding marrow donor centers. In New York and Colorado, minority recruitment was equivalent. In California, Florida, Massachusetts, and New York, the cord blood banks recruited a lower percentage of minorities than those delivering at the respective hospitals. The cord blood banks in California, Colorado, Florida, and Massachusetts recruited a lower percentage of minorities compared with delivery data than the corresponding marrow donor centers compared with census population (p < 0.001). In New York, the percentages were similar. CONCLUSION: The problem of insufficient minority recruitment of cord blood has not yet been solved. Better strategies are needed to recruit minority donors.

A male with sickle SC disease presented at age 8 years with proliferative sickle cell retinopathy (PSCR) and bilateral vitreous hemorrhage which spontaneously resolved, then recurred at 13 years of age. Despite conventional therapy with repeated pan-retinal photocoagulation and pars plana vitrectomy, he developed progressive PSCR and recurrent vitreous hemorrhage over the next 30 months. We describe the successful use of chronic red cell exchange transfusion (RCE) to preserve his vision and stabilize the retinopathy.

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.

Hydroxyethyl starch (hetastarch) is a synthetic glucose compound with extensive clinical use as a volume expander. Because of its red blood cell-sedimenting properties, hetastarch plays a major role in preparation of granulocyte products. Recent concerns have been raised about the use of hetastarch in critically ill patients for the development of renal injury and other severe adverse events. In contrast, granulocyte donors receive much less of this compound during collection procedures, and over many years, minimal toxicity has been documented in these individuals. Furthermore, granulocyte products contain very little hetastarch, and ill effects on renal function have not been associated with their administration. This review assesses available information about the toxicity profile of hetastarch in critically ill patients requiring a volume expander as well as granulocyte donors and recipients. Because of the lack of toxicity in these latter two groups, hetastarch should be available for preparation of granulocyte products and their administration.

BACKGROUND: Granulocyte transfusion may be used in neutropenic patients with severe bacterial or fungal infections that are unresponsive to antibiotic therapy. However, the inability to store granulocyte concentrates limits their clinical usefulness. STUDY DESIGN AND METHODS: Neutrophil chemotaxis and NADPH oxidase activity and the integrity of the neutrophil NADPH oxidase system were examined after apheresis collection and during storage to 48 hours. Neutrophils were mobilized in vivo by G–CSF, collected by apheresis techniques, and stored in apheresis bags in the presence and absence of additional G–CSF. For all experiments, cells were further purified by standard techniques of dextran sedimentation and hypotonic RBC lysis. RESULTS: Neutrophil chemotaxis was preserved to 24 hours of storage but was not affected by the G–CSF added to storage units. The NADPH oxidase system was also preserved as a functioning complex, and both cytosolic proteins and membrane-associated proteins were normal to 48 hours. However, there were divergent responses by intact cells to activating stimuli and reduced oxidase activity in the cell-free system. G–CSF did not appear to significantly affect NADPH oxidase activity or NADPH oxidase system integrity during storage. CONCLUSION: Neutrophils collected after the administration of G–CSF retained functional and biochemical characteristics for at least 24 hours of storage, which suggests additional effects of G–CSF mobilization beyond enhancing PMN yields and the possibility of storage of these components after collection.

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.

A fetus homozygous for alpha-thalassemia-1 was given haploidentical paternal CD34 cells at 13, 19 and 24 weeks' gestation and supported through pregnancy by blood transfusion. The fetal hematocrit ranged between 27 and 47% and between one half and three quarters of this hemoglobin was of recipient (Bart's) type. Intrauterine growth proceeded normally and no significant fetal hydrops was detected. Tests for donor HLA antigens, and alpha-globin DNA, were negative on fetal blood samples drawn before birth. A positive signal for alpha-globin DNA was obtained from cord blood and from marrow obtained at 3 months of age, suggesting that some donor stem cells had persisted in the recipient. The infant's blood mononuclear cells showed little proliferative and no cytotoxic response to the donor while responses to a third party were present. Additional paternal CD34 cells given at 3 months age did not reduce transfusion dependency in the subsequent 6 months. Our results show that repeated transfusions can support an alpha-thalassemia-1 fetus through pregnancy, in this instance without significant birth defects or apparent hypoxic tissue injury. The donor stem cells did not have a survival advantage compared with endogenous stem cells, but appeared to survive in the recipient as judged by the persistence of an alpha-globin DNA signal. In vitro studies of alloreactivity suggest tolerization of the host to the donor's MHC disparity. Future efforts will focus on exploiting this tolerance to improve the level of donor chimerism.

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.

Peroxiredoxins (Prdxs) are a family of proteins which catalyze the reduction of H2O2 through the interaction of active site cysteine residues. Conserved within all plant and animal kingdoms, the function of these proteins is related to protection from oxidation or participation of signaling through degradation of H2O2. Peroxiredoxin 6 (Prdx6), a protein belonging to the class of 1-cys Prdxs, was identified in polymorphonuclear leukocytes or neutrophils, defined by amino acid sequence and activity, and found associated with a component of the NADPH oxidase (Nox2), p67(phox). Prdx6 plays an important role in neutrophil function and supports the optimal activity of Nox2. In this chapter, methods are described for determining the Prdx activity of Prdx6. In addition, the approach for assessing the effect of Prdx6 on Nox2 in the SDS-activated, cell-free system of NADPH oxidase activity is presented. Finally, the techniques for suppressing Prdx6 expression in phox-competent K562 cells and cultured myeloid cells with siRNA and shRNA methods are described. With these approaches, the role of Prdx6 in Nox2 activity can be explored with intact cells. The biochemical mechanisms of the Prdx6 effect on the NADPH oxidase can be investigated with the experimental strategies described.