ALBERT

Description: 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.

Description: 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 

Description: BACKGROUND: Apheresis platelet collection from healthy normal blood donors can reduce the donor peripheral blood platelet concentration by 50% or more. The kinetics of peripheral blood platelet count (PLT) recovery in the apheresis donors over the first 24 hours has not been described. The objective of this study was to determine the recovery kinetics of the donor peripheral blood platelet count following apheresis platelet donation. METHODS: Healthy apheresis platelet donors were enrolled following informed consent. The apheresis platelet collection was performed using the Gambro Trima system (Gambro BCT, Lakewood, CO) following local SOP and manufacturer’s directions for use. The minimum predicted post-count was configured in the Trima to no less than 78K plt/μL. PLT was determined pre-procedure (Pre), immediately post procedure (Post), 4–11 h (FU1) and 11–41 h (FU2) post-donation using standard methods. The PLT recovery was evaluated as the increase in PLT following the donation (Delta). The effects of study site, time of sample, and the fraction of platelets collected (Fpc) at donation on Delta were evaluated using a random effects generalized linear regression model. A full regression model of Delta as a function of study site, follow-up period and Fpc with all main and interaction effects was used to test hypotheses. RESULTS: 548 subjects were entered into the study at 3 study sites; Pre-PLT 276±59 × 103 plt/μL, Post-PLT 205±47 × 103 plt/μL, Fpc 25±10%. No adverse events were reported by any subjects. Recovery of platelet count following apheresis platelet donation is variable between subjects; and the independent variables of study site, follow-period and Fpc accounted for 25% of the total variation in Delta. PLT increased 12.4±0.9 × 103 plt/μL by the time of follow-up sampling (p=0.01), although there was no difference between PLT at FU1 (214±49 × 103 plt/μL) and FU2 (212±47 × 103 plt/μL; p=0.15). None of the donors reached their pre-donation platelet count during the follow-up period. There was no difference in Delta between centers (p=0.23). Fpc had a significant affect on Delta (p

Description: Introduction: TRALI is acute lung injury occurring during or within hours of a blood transfusion. The etiology is thought to be infusion of leukocyte antibodies or neutrophil priming and activation caused by biologically active lipids in blood components. We report a TRALI reaction associated with fresh frozen plasma (FFP) and activation of complement in both the unit of FFP and the patient at the time of the reaction. Case History: A 59 year old male with factor XI was admitted to the hospital with hematochezia and given 3 units of FFP. During infusion of the third unit, he developed dyspnea and cyanosis requiring ventilator and O2 support. A chest x-ray showed bilateral diffuse pulmonary infiltrates, CVP was 3 mm Hg, and an echocardiogram was normal. The symptoms resolved in 3 days. Methods: Samples from donors and/or units were screened for the presence of HLA antibodies by ELISA and lymphocytotoxicity and antibodies detected were typed for HLA specificity and antibody class. Reactivity was determined by flow crossmatch. Serologic and molecular HLA typing was completed on donor and patient samples. Priming activity of the implicated FFP, fresh plasma from donor and recipient, and plasma from controls was completed against freshly isolated neutrophils from the three sources. Significant activity was defined as 〉1.5 times the fMLP stimulated superoxide anion (O2−) production. C3aLE, C4aLE, SC5b-9, and Bb were determined by standard techniques. Results: HLA antibodies were only detected in the third unit of FFP. Samples from this unit and the donor exhibited HLA Class I and II reactivity by ELISA but not lymphocytotoxicity. Flow crossmatch cells demonstrated Class II, IgG reactivity of donor serum against recipient DR11, 13. No autologous reactivity was demonstrated. The FFP unit primed the fMLP response in donor, recipient and control neutrophils 2.6, 3.1, and 3.4 fold above baseline. Testing of donor, recipient and control plasma obtained 3 months after the reaction showed no priming against the same battery of cells (priming ratio 0.8–1.3). C4aLE (105%, control range 24–176%); C3aLE (476%, control range 21–180%); and Bb (351%, control range 31–169%) were elevated in recipient samples obtained during the TRALI reaction and SC5b-9 was at the high end of normal (164%, control 0–200%). These returned to normal after the reaction. Strikingly, evidence of complement activation was seen in the FFP unit (C4aLE 214%, C3aLE 402%, C5b-9 213%) but not in subsequent samples from the donor. Conclusion: These studies document a TRALI reaction with symptoms expressed during the administration of FFP. One unit exhibited HLA Class I and II antibodies, the latter of which bound to the recipient’s cells. Priming activity was seen with plasma from the implicated unit, not in subsequent samples from the donor. Laboratory studies document activation of complement in the FFP infused but not donor samples. Plasma from the recipient at the time of the reaction also exhibit activation of complement which became normal after the TRALI resolved. Infusion of the FFP with activated complement capable of priming neutrophils may have induced pulmonary leukostasis and TRALI quite distinct from any subsequent effect of antibodies. Although the cause of FFP complement activation is not defined, these results suggest alternative mechanisms involving complement may be responsible for HLA antibody-associated TRALI.

Description: 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.

Description: Introduction: Production of reactive oxygen species through a respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of the components of the NADPH oxidase enzyme system (p47phox, p67phox, p40phox, gp91phox, p22phox and Rac) and expression of the activity of the system to produce superoxide anion (O2 −). We recently identified a neutrophil protein with an approximate MW of 29 kDa which binds to the p67phox, is classified as a peroxiredoxin (Prx) and translocates to the plasma membrane during stimulation of the neutrophil. Additional studies demonstrate that this protein (p29 Prx) increases production of O2 − in a cell-free system of oxidase activity in a specific, stoichiometric manner and that the cysteine residues of p29 Prx, at amino acid positions 47 and 91, are required for this activity. The current studies demonstrate the role of p29 Prx in oxidase activity using the technique of small interfering RNA (siRNA) to degrade specific mRNA and decrease the expression of the protein. Methods: siRNA probes for p29 Prx were constructed based on standard constraints for unique 19 nucleotide binding sites along with other sequence selection criteria. Six probes were constructed based on the cDNA sequence of p29 Prx; one resulted in significant knockdown of p29 Prx. Inactive siRNA fluorescently labeled was obtained commercially. K562 cells, stably transfected with the p67phox, p47phox, gp91phox and low affinity fMLP receptor, were cultured under standard conditions and expressed p29 Prx message by RT-PCR and protein by Western blot. Transgenic K562 cells were transfected with siRNA or GFP labeled control siRNA with Nucleofector technology. Cell counts and viability were determined by standard techniques. For Western blots, proteins from cell lysates were separated on 10% SDS-PAGE and blotted onto nitrocellulose, and specific proteins were detected with polyclonal antibodies to p29 Prx, actin or p67phox, p47phox, gp91phox, and p22phox by chemiluminescent technique. Results: After transfection with the active siRNA for p29 Prx, inactive or labeled siRNA, the K562 cells were harvested at 24, 48, and 72 hours. A knockdown by one of the 6 siRNAs resulted in decreased levels of p29 Prx by Western blot. Optimum knockdown was achieved by transfection of 6 μg siRNA and the decrease in p29 Prx observed at 24 hours but was optimum after 48 hours. Under these conditions, a decrease in p29 Prx by 50–60% detected by Western blot was achieved with no differences in levels of actin or any of the phox proteins. The viability of control cells and siRNA transfected cells was not different. Cells transfected with siRNA for p29 Prx which demonstrated a knockdown of this protein exhibited decreased respiratory burst (O2 −) in response to fMLP (measured by chemiluminescence) or PMA (cytochrome c reduction) compared to control porated or transfected cells. Conclusion: These results correlate with in vitro studies of recombinant p29 Prx in the SDS cell-free system of oxidase activation. Decreased levels of p29 Prx result in decreased oxidase activity on transgenic K562 cells. p29 Prx may be important for the expression of the oxidase enzyme system through its antioxidant or signaling activity.

Description: 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.

Description: 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.

Description: Abstract 1354 Poster Board I-377 Introduction We have identified a 29 kDa protein from human neutrophils which binds to the NADPH oxidase component p67phox and enhances superoxide anion (O2−) production in a cell-free, reconstituted, NADPH oxidase system. The protein was identified as peroxiredoxin VI by sequence and the recombinant molecule was found to have both peroxiredoxin activity and calcium-independent PLA2 activity that is optimum at low pH (aiPLA2). Although p29 Prdx VI is found in many tissues, its role in myeloid cells is not well established. To explore other roles of p29, in addition to its effect on the respiratory burst, a PLB-985 cell line with shRNA mediated knockdown of p29 Prdx VI was established. Chemotaxis, as well as ingestion and killing S. aureus were determined in knockdown and control cells. Methods PLB-985 cells were transfected with a plasmid encoding a p29 Prdx VI targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx VI was confirmed by Western blot with no changes in actin or other oxidase components. After maturation of the knockdown and control cells by DMSO for 4 days, each was combined with serum opsonized Staph. aureus in a 2 to1 human cell to bacterial cell ratio, bacterial cells remaining at various times were measured by plating aliquots of the cell mixtures and counting bacterial colonies which grew overnight. To evaluate ingestion, aliquots of the cell mixtures were transferred to slides by cytospin, stained, and examined under a light microscope to determine what proportion of PLB-985 cells had internalized bacteria. To evaluate chemotaxis, distances of migration toward chemo-attractant (opsonized zymosan) in a Boyden chamber were measured for differentiated p29 Prdx VI knockdown and control cells. Results Using stable expression of shRNA p29 protein was reduced to 31+/-18% (SD) of that in non-knockdown control cells. In two separate assays of bactericidal activity, cells without knockdown of p29 Prdx VI had 17 and 13% of initial bacteria surviving at 30 min; cells with p29 Prdx VI knockdown had 30 and 56% of bacteria surviving. This defect in bactericidal activity since ingestion was no different between the two types of cells at 0, 5, 10, and 15 min after addition of the bacteria. In response to zymosan activated serum, stimulated directed migration (distance of leading front in response to zymosan activated serum minus distance of leading front in response to buffer) was greater in cells without knockdown (23.9 ± 3.0 microns, mean ± SEM, n = 4 separate experiments) than movement by cells with knockdown of p29 Prdx VI (18.3 ± 5.3 microns). The difference was significant, p