Oxidase Activity In Neutrophils Research Articles

#2950 IGA NEPHROPATHY: PUTTING THE A IN THE UNACQUAINTED

Abstract Background and Aims IgA nephropathy (IgAN) is a common glomerulonephritis. Rarely it can be associated with other diseases and screening for these associations can be missed. We present 2 cases diagnosed as IgAN which were associated with rare underlying conditions. The first (patient X) is a 51-year-old female, who presented with new onset haematoproteinuria on a background of inflammatory arthritis, interstitial lung disease (ILD) and a recurrent maculopapular rash. The second case (patient Y) is a 34-year-old male, who presented with unexplained rapidly progressive chronic kidney disease and proteinuria. He had a background of hidradenitis suppurativa, acne rosacea, Crohn's disease and recurrent infections. Method X's immunology was negative aside from a weakly positive ANA and p-ANCA but negative ELISA. Her myositis panel was positive for PM/ScL antibody. Imaging showed multifocal ground glass opacification, consistent with ILD. Her renal function was normal, but renal histology demonstrated IgAN, without evidence of vasculitis. With these findings in mind, a diagnosis of anti-synthetase syndrome (AS) with IgAN was proposed. Y, conversely, had abnormal renal function, with significant proteinuria. Immunology screen was unremarkable other than a raised serum IgA, weakly positive MPO and equivocal PR3. Renal biopsy showed IgAN with severe interstitial fibrosis and tubular atrophy, and over 50% sclerosed glomeruli. Y later developed haemolytic anaemia; his neutrophil oxidase activity was absent, raising the possible diagnosis of chronic granulomatous disease (CGD). This was subsequently confirmed with genetic testing demonstrating an autosomal recessive gene for CGD. Results Both X's proteinuria and symptomatic burden significantly improved with methotrexate. Y was commenced on antibiotics and antifungals in light of the heightened risk of infection which CGD carries. He is now on maintenance haemodialysis. The only curative treatment for CGD is allogenic hematopoietic stem cell transplantation from a matched donor, for which he is being considered. Conclusion AS is a poorly defined, rare syndrome characterised clinically by ILD, arthritis and myositis. It is immunologically characterised by autoantibodies against aminoacyl tRNA synthetases; whilst X did not test positive, this does not preclude AS, as autoantibody titres fluctuate depending on disease activity [1]. Renal involvement in AS is extremely rare, or so the data would suggest. Couvrat-Desvergnes et al. examined 14 biopsies of patients with inflammatory myopathies plus renal involvement. Only 3 had AS; of these, 2 had IgAN. Other cases of IgA are described only in case reports. In all cases, immunosuppressive treatments were effective in achieving remission. Some cases describe other renal implications in AS, namely TMA and amyloidosis (Table 1). CGD is a rare primary immunodeficiency caused by genetic defects in the NADPH oxidase complex, which is critical in the destruction of phagocytes. Consequently, patients have recurrent infections and abnormal inflammatory responses result in granuloma formation. The incidence of renal disease in CGD has been suggested by some papers as being as low as 2.7% [2]. From review of the other cases of CGD with associated IgAN, it seems the patient we present is the oldest (Table 2). Interestingly, only 1 other case reports glomerulonephritis as the presenting complaint for CGD [3]. These cases highlight that, while IgAN is a disease in its own right, its diagnosis may shine light on other associated pathologies. It is crucial for nephrologists to be aware of rare associations as often it has impact on further management.

Lentiviral gene therapy for X-linked chronic granulomatous disease recapitulates endogenous CYBB regulation and expression

AbstractX-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.

New Insights on NETosis Induced by Entamoeba histolytica: Dependence on ROS from Amoebas and Extracellular MPO Activity.

NETosis is a neutrophil process involving sequential steps from pathogen detection to the release of DNA harboring antimicrobial proteins, including the central generation of NADPH oxidase dependent or independent ROS. Previously, we reported that NETosis triggered by Entamoeba histolytica trophozoites is independent of NADPH oxidase activity in neutrophils, but dependent on the viability of the parasites and no ROS source was identified. Here, we explored the possibility that E. histolytica trophozoites serve as the ROS source for NETosis. NET quantitation was performed using SYTOX® Green assay in the presence of selective inhibitors and scavengers. We observed that respiratory burst in neutrophils was inhibited by trophozoites in a dose dependent manner. Mitochondrial ROS was not also necessary, as the mitochondrial scavenger mitoTEMPO did not affect the process. Surprisingly, ROS-deficient amoebas obtained by pre-treatment with pyrocatechol were less likely to induce NETs. Additionally, we detected the presence of MPO on the cell surface of trophozoites after the interaction with neutrophils and found that luminol and isoluminol, intracellular and extracellular scavengers for MPO derived ROS reduced the amount of NET triggered by amoebas. These data suggest that ROS generated by trophozoites and processed by the extracellular MPO during the contact with neutrophils are required for E. histolytica induced NETosis.

Clinical, functional and genetic characterization of 16 patients suffering from chronic granulomatous disease variants-identification of 11 novel mutations in CYBB.

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The most common form is the X-linked CGD (X91-CGD), caused by mutations in the CYBB gene. Clinical, functional and genetic characterizations of 16 CGD cases of male patients and their relatives were performed. We classified them as suffering from different variants of CGD (X910 , X91- or X91+ ), according to NADPH oxidase 2 (NOX2) expression and NADPH oxidase activity in neutrophils. Eleven mutations were novel (nine X910 -CGD and two X91- -CGD). One X910 -CGD was due to a new and extremely rare double missense mutation Thr208Arg-Thr503Ile. We investigated the pathological impact of each single mutation using stable transfection of each mutated cDNA in the NOX2 knock-out PLB-985 cell line. Both mutations leading to X91- -CGD were also novel; one deletion, c.-67delT, was localized in the promoter region of CYBB; the second c.253-1879A>G mutation activates a splicing donor site, which unveils a cryptic acceptor site leading to the inclusion of a 124-nucleotide pseudo-exon between exons 3 and 4 and responsible for the partial loss of NOX2 expression. Both X91- -CGD mutations were characterized by a low cytochrome b558 expression and a faint NADPH oxidase activity. The functional impact of new missense mutations is discussed in the context of a new three-dimensional model of the dehydrogenase domain of NOX2. Our study demonstrates that low NADPH oxidase activity found in both X91- -CGD patients correlates with mild clinical forms of CGD, whereas X910 -CGD and X91+ -CGD cases remain the most clinically severe forms.

DPI Selectively Inhibits Intracellular NADPH Oxidase Activity in Human Neutrophils.

Neutrophils are capable of producing significant amounts of reactive oxygen species (ROS) by the phagocyte NADPH oxidase, which consists of membrane-bound and cytoplasmic subunits that assemble during activation. Neutrophils harbor two distinct pools of the membrane-localized oxidase components, one expressed in the plasma membrane and one in the membranes of intracellular granules. Assembly of active oxidase at either type of membrane leads to release of extracellular ROS or to the production of ROS inside intracellular compartments, respectively. The cytoplasmic NADPH oxidase subunit p40phox seems selectively critical for the ability to generate intracellular ROS, and the recent characterization of patients with p40phox deficiency implies that selective loss of intracellular neutrophil ROS leads to disease with pronounced hyperinflammatory features, suggesting that these ROS are critical for regulation of inflammation. This study aimed at characterizing two pharmacological NADPH oxidase inhibitors, the newly described GSK2795039 and the widely used diphenyleneiodonium (DPI), focusing on their abilities to inhibit human neutrophil ROS production extra- and intracellularly. Whereas GSK2795039 blocked extra- and intracellular NADPH oxidase activity equally, DPI was found to selectively interfere with intracellular ROS production. Selectivity for the intracellular NADPH oxidase was evident as a lower IC50 value, faster onset, and irreversibility of inhibition. We found no evidence of direct interactions between DPI and p40phox, but the selectivity of DPI confirms that regulation of NADPH oxidase activity in neutrophils differs depending on the subcellular localization of the enzyme. This information may be used to pharmacologically mimic p40phox deficiency and to further our understanding of how intracellular ROS contribute to health and disease.

Sleep deprivation alters neutrophil functions and levels of Th1-related chemokines and CD4+ T cells in the blood.

The state of knowledge about the effect of sleep deprivation on the immune system is scarce and conflicting. It would therefore be useful to investigate the consequences of sleep deprivation on the immune system. We have studied the effect of sleep deprivation on the changes in neutrophil functions, and the ex vivo proliferative pattern of CD4+ T lymphocytes in relationship with blood cytokine and chemokine levels due to the crucial role of these cells in mounting potent immune responses. Healthy volunteers were followed for 3weeks. They had normal sleep in weeks 1 and 3 and they were sleep-deprived on week 2, sleeping < 6h per 24h, a pattern similar to sleep behaviors of many chronically sleep-deprived individuals. We assessed the levels of Th1/Th2 and inflammatory cytokines and chemokines, CD4+ T cells, and the NADPH oxidase activation and phagocytic functions in neutrophils. Our results suggest that sleep deprivation leads to a decreased neutrophil capacity to phagocytose bacteria and activate NADPH oxidase (p < 0.05). Sleep deprivation was associated with a potential increase in CXCL9 levels and decrease in CXCL10/CXCL9 and CCL5/CXCL9 ratios (p < 0.05). Furthermore, our results suggest that the decrease in CD4+ T cell due to sleep deprivation was not associated with changes in their proliferation as observed by Ki67 levels, but rather, it correlated with changes in CXCL10/CXCL9 ratio (p < 0.05). Sleep deprivation may lead to a decreased phagocytosis and NADPH oxidase activity in neutrophils and a decrease in the levels of CD4+ T cells which is related to changes in the Th1-related chemokine balance.

Neutrophil-dependent pentraxin-3 and reactive oxygen species production modulate endothelial dysfunction in haemodialysis patients.

The aim of this study was to investigate neutrophil activation and its role in long pentraxin-3 (PTX3) release and oxidative stress generation during haemodialysis (HD) and to correlate neutrophil PTX3 and oxidant expression with endothelial dysfunction. Forty-seven uraemic patients on stable HD, 12 healthy subjects and 15 patients with congestive heart failure (New York Heart Association classes III and IV) were enrolled. Neutrophil PTX3 protein expression was evaluated by confocal microscopy. l -selectin expression, intracellular PTX3 localization and reactive oxygen species (ROS) generation in human neutrophils were measured by flow cytometry. NADPH-dependent superoxide generation was investigated by chemiluminescence. PTX3 plasma concentrations were measured by ELISA. Endothelial dysfunction was studied by flow-mediated dilation (FMD). The low baseline levels of FMD significantly improved after HD, but worsened by 24 h. A significant up-regulation of PTX3 protein expression, localized within secondary granules, was detected in neutrophils isolated at 30 and 240 min of HD, along with an increase in l -selectin expression. The up-regulation in intracellular PTX3 in neutrophils was associated with a significant increase in PTX3 plasma concentrations at 240 min. HD increased ROS production and NADPH oxidase activity in neutrophils. In a univariate analysis, pre-treatment with FMD was inversely correlated with PTX3 expression and ROS generation in neutrophils. In a multivariate analysis, both circulating pre-HD PTX3 and intracellular ROS generation by neutrophils were independent predictors of abnormal FMD. Neutrophil overexpression of PTX3 is associated with ROS overproduction and endothelial dysfunction and may represent an emerging marker of vascular damage progression in HD patients.

FMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils.

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP), diphenyleneiodonium (DPI), and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor) inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

PS-303 Vitamin D Increases Roi Production In Term And Preterm Infants: Possible Mechanism Of Enhanced Antibacterial Effect

Introduction Newborn infants are at risk of vitamin D deficiency and various studies implicate vitamin D as having immunomodulatory effect. Adequate generation of reactive oxygen intermediates (ROI) by neutrophils (PMN) during sepsis is bactericidal. However, production of neutrophil oxidase activity in the presence of sepsis is impaired in neonates. Aim To examine the in vitro effect of 1, 25(OH) 2 D 3 on whole blood PMN and monocyte ROI, TLR4, CD11b in newborn infants during sepsis in vitro . Methods Whole blood from preterm infants in vitro sepsis) and 1,25(OH) 2 D 3 . Results ROI production from preterm and term neonatal neutrophils incubated with LPS alone was not significantly increased in contrast to adults. However pre-incubation with 1,25(OH) 2 D 3 before adding LPS demonstrated a significant increase (p = 0.001) in ROI production for both preterm and term infants while simultaneous LPS and 1,25(OH)2D3 had no effect. Conclusion New born infants were hypo-responsive in the presence of sepsis in vitro which recovered on pre-treatment with 1, 25(OH) 2 D 3 . Pre-treatment with vitamin D may improve term and preterm infants’ antibacterial responses.

Transducible form of p47phox and p67phox compensate for defective NADPH oxidase activity in neutrophils of patients with chronic granulomatous disease

Protein delivery to primary cells by protein transduction domain (PTD) serves as a novel measure for manipulation of the cells for biological study and for the treatment of various human conditions. Although the method has been employed to modulate cellular function in vitro, only limited reports are available on its application in the replacement of deficient signaling molecules into primary cells. We examined the potential of recombinant proteins to compensate for defective cytosolic components of the NADPH oxidase complex in chronic granulomatous disease (CGD) neutrophils in both p47phox and p67phox deficiency. The p47phox or p67phox protein linked to Hph-1 PTD was effectively expressed in soluble form and transduced into human neutrophils efficiently without eliciting unwanted signal transduction or apoptosis. The delivered protein was stable for more than 24h, expressed in the cytoplasm, translocated to the membrane fraction upon activation, and, most importantly able to restored reactive oxygen species (ROS) production. Although research on human primary neutrophils using the protein delivery system is still limited, our data show that the protein transduction approach for neutrophils may be applicable to the control of local infections in CGD patients by direct delivery of the protein product.

Cesium cessation? An advantage of pathogen reduction treatments

Cesium cessation? An advantage of pathogen reduction treatments

Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADPH oxidase activity in neutrophils

In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast damped Ca2+ spikes with a period of 15s and slower irregular spikes with a period greater than 50s. Spikes in Ca2+ occurred in the absence of Ca2+ influx, but the amplitude was damped by inhibition of Ca2+ influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production, we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca2+ but, despite subsequent spikes in Ca2+ concentration, no oscillations in oxidase activity could be detected. ATP induced spikes in Ca2+ in a very reproducible way and we propose that the Ca2+ signal is an on-switch for oxidase activity, but the activity is apparently not directly correlated with spiking activity in cytosolic Ca2+.

Chronic Granulomatous Disease: Lessons from a Rare Disorder

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency with X-linked or autosomal recessive inheritance involving defects in genes encoding phox proteins, which are the subunits of the phagocyte NADPH oxidase. This results in failure to produce superoxide anion and downstream antimicrobial oxidant metabolites and to activate antimicrobial proteases. Affected patients are susceptible to severe, life-threatening bacterial and fungal infections and excessive inflammation characterized by granulomatous enteritis resembling Crohn's disease and genitourinary obstruction. Early diagnosis of CGD and rapid treatment of infections are critical. Prophylaxis with antibacterial and mold-active antifungal agents and the administration of interferon-γ has significantly improved the natural history of CGD. Currently, the only cure is allogeneic hematopoietic cell transplant (HCT), although there remains controversy as to which patients with CGD should get a transplant. Allele-based HLA typing of alternative donors, improved supportive care measures, and use of reduced toxicity conditioning have resulted in event-free survival (EFS) of at least 80% even with an unrelated donor and even better when the patient has no active infections/inflammation. Gene correction of CGD would eliminate the risks of graft-versus-host disease (GVHD) and the immunoablative chemotherapy required for allogeneic HCT. Based on gene therapy trials in patients with SCID-X1, ADA-SCID, and the early experience with CGD, it is clear that at least some degree of myeloablation will be necessary for CGD as there is no inherent selective growth advantage for gene-corrected cells. Current efforts for gene therapy focus on use of lentivector constructs, which are thought to be safer from the standpoint of insertional mutagenesis and more efficient in transducing hematopoietic stem cells (HSCs).

Generation of neutrophil priming activity by cell‐containing blood components treated with pathogen reduction technology and stored in platelet additive solutions

Storage of cell-containing blood components such as platelet concentrates (PCs) and red blood cells (RBCs) results in generation of biologically active compounds, many of which may be associated with adverse transfusion events. Priming of the neutrophil oxidase activity is a common characteristic of many of the biologically active compounds found in stored blood. We evaluated the priming activity of pathogen reduction technology (PRT)-treated PCs stored in plasma or platelet additive solution (PAS) and PRT-treated RBCs. PCs were collected with Trima or Amicus equipment and were PRT treated with the Mirasol PRT system or the Intercept Blood System. Some units were gamma irradiated. Products were stored in 100% plasma or 35% plasma plus PAS. RBCs were washed and PRT treated before storage. Samples were removed throughout storage and priming of the oxidase activity was measured. Platelets collected on Trima or Amicus equipment and stored in plasma or PAS demonstrated increasing priming activity during 5 to 7 days of storage. Gamma irradiation, but not PRT treatment with either technology, further enhanced this priming activity. Supernatants of RBCs stored for 42 days induced priming in untreated controls, but not in washed or Mirasol PRT-treated test products. Production of oxidase priming activity increased during storage in all blood products. No significant differences were associated with the collection method, storage in PAS, or PRT treatment. The generation of biologically active compounds, which may serve as an etiology for adverse events, appears to be independent of these processes for collection, storage, and pathogen reduction.

Rapid Genetic Analysis of X-Linked Chronic Granulomatous Disease by High-Resolution Melting

High-resolution melting analysis was applied to X-linked chronic granulomatous disease, a rare disorder resulting from mutations in CYBB. Melting curves of the 13 PCR products bracketing CYBB exons were predicted by Poland's algorithm and compared with observed curves from 96 normal individuals. Primer plates were prepared robotically in batches and dried, greatly simplifying the 3- to 6-hour workflow that included DNA isolation, PCR, melting, and cycle sequencing of any positive products. Small point mutations or insertions/deletions were detected by mixing the hemizygous male DNA with normal male DNA to produce artificial heterozygotes, whereas detection of gross deletions was performed on unmixed samples. Eighteen validation samples and 22 clinical kindreds were analyzed for CYBB mutations. All blinded validation samples were correctly identified. The clinical probands were identified after screening for neutrophil oxidase activity. Nineteen different mutations were found, including seven near intron-exon boundaries predicting splicing defects, five substitutions within exons, three small deletions predicting premature termination, and four gross deletions of multiple exons. Ten novel mutations were found, including (c.) two missense (730T>A, 134T>G), one nonsense (90C>A), four splice site defects (45 + 1G>T, 674 + 4A>G, 1461 + 2delT, and 1462-2A>C), two small deletions (636delT, 1661_1662delCT), and one gross deletion of exons 6 to 8. High-resolution melting can provide timely diagnosis at low cost for effective clinical management of rare, genetic primary immunodeficiency disorders.

Chronic granulomatous disease (CGD) due to a novel mutation in CYBB

Aim A previously healthy 2-year-old boy presented with a 2-month history of lymphadenitis over his right popliteal fossa and inguinal regions. Burkholderia cepacia was isolated from his post-surgical drainage. One month later he developed persistent purulent discharge from his popliteal fossa surgical wound and recurrence of inguinal lymphadenitis. Laboratory investigation for CGD was performed. Method Neutrophil oxidase activity was assessed by nitroblue tetrazolium (NBT) slide and dihydrorhodamine (DHR) flow cytometry methods. Expression of phox proteins was measured by Western blot. Mutation analysis of the cytochrome b beta gene (CYBB) was performed by amplifying the coding region from cDNA by PCR and directly sequencing by the dideoxy chain termination method. Results Expression of gp91phox, p47phox and p67phox and translocation of p47phox and p67phox was normal. A novel hemizygous missense mutation causing a C1524A transition, leading to a A488D substitution, was identified in the CYBB gene. Discussion The patient was diagnosed with X-linked CGD with normal gp91-phox expression, due to a missense mutation in CYBB. Establishing a definitive diagnosis of CGD requires a thorough clinical and laboratory workup.

Electrochemistry and chemiluminescence techniques compared in the detection of NADPH oxidase activity in phagocyte cells

Several methodologies have been used in clinical chemistry for real-time assessment of NADPH oxidase primary product superoxide anion which dismutases to hydrogen peroxide. Among these methodologies, isoluminol chemiluminescence (CL) is considered to be one of the more sensitive and reliable techniques for the assessment of NADPH oxidase activity in neutrophils. The electrochemical technique was recently designed and also applied for real-time detection of NADPH oxidase activity in neutrophils but its reliability and sensitivity has not been investigated so far. In this study, isoluminol CL and electrochemical techniques were investigated and compared by monitoring the generation of superoxide and hydrogen peroxide in both PLB 985 cell line differentiated into neutrophil-like cells and human neutrophils. The electrochemical technique was shown to be as sensitive as that of CL and able to detect the reactive oxygen species (ROS) release of as low as 500 cells. Thus, the electrochemical technique could be used as an alternative to optical techniques for the evaluation of extracellular ROS in phagocyte cells.

Complement Activation in Transfusion Related Acute Lung Injury (TRALI).

Background: TRALI is defined as acute lung injury occurring within six hours of a transfusion of blood products. Two major etiologies have been identified and include leukocyte antibodies activating complement. We report two patients with TRALI reactions who had documented activation of complement.Case Studies. Case 1: A 59 year old male with Factor XI deficiency and hematochezia was given 3 units of fresh frozen plasma (FFP). During infusion of the third unit, the patient developed dyspnea and cyanosis requiring ventilator support. A chest x-ray showed new diffuse infiltrates; CVP and an echocardiogram were normal. The symptoms resolved in 3 days.Case 2: An 87 year old female required surgical repair of a hip fracture. Four units were infused and with the last one, the patient developed chills, fever and tachypnea. On 6 l/min of O2, she had a saturation of 58%. Chest x-ray showed bilateral infiltrates; the echocardiogram was normal. The patient sustained a cardiac arrest and could not be resuscitated.Methods: Samples from donors or FFP units were screened for HLA and granulocyte antibodies. Priming of the fMLP stimulated neutrophil oxidase activity was performed by standard assay. Plasma from products implicated in TRALI and plasma samples from patients before and during the TRALI reaction were assayed for priming of the fMLP stimulated respiratory burst in neutrophils; C3a, C4a, C5a, C5–9, and Bb were measured by standard techniques.Results: In Case 1, HLA Class II, IgG reactivity by flow cytometry was documented with plasma from the implicated donor and recipient cells. Priming of neutrophils was detected in the implicated unit as well as the recipient's plasma during the reaction in comparison to pre-transfusion specimen. Complement activation products were increased in samples during the reaction and the product being infused at the onset of TRALI. In the second case, HLA Class II antibodies with a specificity which would interact with the recipient's antigens were detected in the product related to the TRALI reaction. Priming activity of the neutrophil oxidase was documented in the infused product and in post vs. pre reaction patient samples. Complement activation products were increased in the implicated FFP unit and C5a was increased in the patient's post samples. Additional cases are being assessed for complement activation.Conclusions: In these cases of TRALI, the implicated products exhibited priming activity as well as antibodies which reacted with recipient antigens by flow crossmatch or by documentation of the cognate antigen. Complement activation was documented in patient samples during the reaction as opposed to those collected before the transfusion. Most importantly, complement was activated in the product transfused during the onset of TRALI symptoms. Thus, complement activation in the product is the most likely cause of the TRALI reaction. Infusion of products containing activated complement components may provide an alternative mechanism for TRALI reactions.

Neutrophil NADPH-oxidase activation by an annexin AI peptide is transduced by the formyl peptide receptor (FPR), whereas an inhibitory signal is generated independently of the FPR family receptors

Truncation of the N-terminal part of the calcium-regulated and phospholipid-binding protein annexin AI has been shown to change the functional properties of the protein and to generate immunoregulatory peptides. Proinflammatory as well as anti-inflammatory signals are triggered by these peptides, and the two formyl peptide receptor (FPR) family members expressed in neutrophils, FPR and FPR-like 1 (FPRL1), have been suggested to transduce these signals. We now report that an annexin AI peptide (Ac9-25) activates, as well as inhibits, the neutrophil release of superoxide anions. Results obtained from experiments with receptor antagonists/inhibitors, desensitized cells, and transfected cells reveal that the Ac9-25 peptide activates the neutrophil reduced nicotinamide adenine dinucleotide phosphate oxidase through FPR but not through FPRL1. The Ac9-25 peptide also inhibits the oxidase activity in neutrophils triggered, not only by the FPR-specific agonist N-formyl-Met-Leu-Phe but also by several other agonists operating through different G protein-coupled receptors. Our data show that the two signals generated by the Ac9-25 peptide are transmitted through different receptors, the inhibitory signal being transduced by a not-yet identified receptor distinct from FPR and FPRL1.

Cryptic O2- -generating NADPH oxidase in dendritic cells.

All the components of the O(2)(-)-generating NADPH oxidase typically found in neutrophils, namely a membrane-bound low potential flavocytochrome b and oxidase activation factors of cytosolic origin, are immunodetectable in murine dendritic cells (DCs). However, in contrast to neutrophils, DCs challenged with phorbol myristate acetate (PMA) can barely mount a significant respiratory burst. Nevertheless, DCs generate a substantial amount of O(2)(-) in the presence of PMA following preincubation with pro-inflammatory ligands such as lipopolysaccharide and pansorbin, and to a lesser extent with anti-CD40 or polyinosinic polycytidylic acid. We found that the virtual lack of the oxidase response to PMA alone is specifically controlled in DCs. Through the use of homologous and heterologous cell-free systems of oxidase activation, we showed the following: (1) a NADPH oxidase inhibitory factor is located in DC membranes; it exerts its effect on oxidase activation and not on the activated oxidase. (2) The inhibition is relieved by pretreatment of DC membranes with beta-octylglucoside (beta-OG). (3) The beta-OG-extracted inhibitory factor prevents the activation of neutrophil oxidase. (4) The inhibitory activity is lost after treatment of DC membranes with proteinase K or heating, which points to the protein nature of the inhibitory factor. Overall, these data indicate that the O(2)(-)-generating oxidase in DCs is cryptic, owing to the presence of a membrane-bound inhibitor of protein nature that prevents oxidase activation. The inhibition is relieved under specific conditions, including a prolonged contact of DCs with pro-inflammatory ligands from microbial origin, allowing a substantial production of O(2)(-), which may contribute to the response of DCs to a microbial exposure.