A 5-week-old male infant presented with severe bacterial infections and poor wound healing, suggesting a neutrophil defect. Neutrophils from this patient exhibited decreased chemotaxis, polarization, ...azurophilic granule secretion, and superoxide anion (O2
-) production but had normal expression and up-regulation of CD11b. Rac2, which constitutes >96% of the Rac in neutrophils, is a member of the Rho family of GTPases that regulates the actin cytoskeleton and O2
-production. Western blot analysis of lysates from patient neutrophils demonstrated decreased levels of Rac2 protein. Addition of recombinant Rac to extracts of the patient neutrophils reconstituted O2
-production in an in vitro assay system. Molecular analysis identified a point mutation in one allele of the Rac2 gene resulting in the substitution of Asp57 by an Asn (Rac2D57N). Asp57 is invariant in all defined GTP-binding proteins. Rac2D57Nbinds GDP but not GTP and inhibits oxidase activation and O2
-production in vitro. These data represent the description of an inhibitory mutation in a member of the Rho family of GTPases associated with a human immunodeficiency syndrome.
Rho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A ...patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G→A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways.
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 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 < .0004) and patients with cardiac disease (P < .0006). TRALI was associated with older platelets (P = .014). In the prospective study, antileukocyte antibodies were found in only 3.6% of cases. The implicated blood components had greater PMN priming activity than controls (P < .05), and compared with pretransfusion samples, TRALI patients' plasma demonstrated increases in both interleukin 6 (IL-6) and lipid (neutral lipids and lysophosphatidylcholines) priming activity (P < .05). We conclude that TRALI may be more frequent than previously recognized and that patient susceptibility, product age, and increased levels of bioactive lipids in components may predispose patients to TRALI. TRALI, like the acute respiratory distress syndrome, may be a 2-event phenomenon with both recipient predisposition and factors in the stored units playing major roles.
Background
Red blood cell (RBC) transfusion is a life‐saving intervention for critically ill patients; however, it has been linked to increased morbidity and mortality. We hypothesize that a number ...of important proteins accumulate during routine storage of RBCs, which may explain some of the adverse effects seen in transfused patients.
Study Design
Five RBC units were drawn and divided (half prestorage leucoreduced (LR‐RBC) and half left as an unmodified control (RBC). The supernatant was separated on days 1 and 42 of storage and proteomic analyses completed with in‐gel tryptic digestion and nano‐liquid chromatography tandem mass spectrometry.
Results
In RBC supernatants, 401 proteins were identified: 203 increased with storage, 114 decreased, and 84 were unchanged. In LR‐RBC supernatant, 231 proteins were identified: 84 increased with storage, 30 decreased, and 117 were unchanged. Prestorage leucoreduction removed many platelet‐ and leucocyte‐derived structural proteins; however, a number of intracellular proteins accumulated including peroxiredoxins (Prdx) 6 and latexin. The increases were confirmed by immunoblotting, including the T‐phosphorylation of Prdx‐6, indicating that it may be functioning as an active phospholipase. Active matrix metalloproteinase‐9 also increased with a coinciding decrease in the metalloproteinase inhibitor 1 and cystatin C.
Conclusion
We conclude that a number of proteins increase with RBC storage, which is partially ameliorated with leucoreduction, and transfusion of stored RBCs may introduce mediators that result in adverse events in the transfused host.
BACKGROUND: Transfusion‐related acute lung injury (TRALI) is clinically similar to the adult respiratory distress syndrome (ARDS) and has been linked to the transfusion of leukocyte antibodies in ...blood components. Animal model have implicated neutrophil (PMN)‐priming agents in ARDS; however, two agents were required. Previous studies showed the generation of PMN‐priming agents during blood storage. Thus the association of PMN‐priming agents with TRALI was examined. STUDY DESIGN AND METHODS: Ten patients with TRALI and 10 with febrile or urticarial reactions (control group) were evaluated. The presence of PMN‐priming activity was tested in the patients' pretransfusion and posttransfusion blood samples by incubating PMNs with these samples followed by activation of the respiratory burst. Plasma lipids were separated by normal‐phase high‐performance liquid chromatography (HPLC), and the priming activity was evaluated. The presence of leukocyte antibodies was determined in the blood donors and patients with TRALI. RESULTS: Significantly more PMN‐priming activity was present in the posttransfusion sera (11.4 +/− 1.8 nmol superoxide anion/min, mean +/− SEM; n=10) and plasma of patients with TRALI than in their pretransfusion sera (6.5 +/− 1.5: n=10) or in the pretransfusion and posttransfusion sera (5.1 +/− 1.3, n=10; and 4.5 +/− 1.4, n=10, respectively) and from the controls (p < 0.05). HPLC separation of lipids demonstrated that three active species were present in the posttransfusion plasma samples of TRALI patients. All the patients with TRALI had underlying clinical factors, such as infection, cytokine administration, recent surgery, or massive transfusion, while only 2 of 10 control patients had these clinical conditions. None of the donors had significant titers of HLA or HLA‐DR antibodies; however, 50 percent had weak positivity for granulocyte antibodies. CONCLUSION: TRALI is the result of two clinical events, the first being a predisposing clinical condition and the second being the transfusion of biologically active lipids in stored blood.
Transfusion-related acute lung injury (TRALI) is a serious complication of hemotherapy. During blood storage, lipids are generated and released into the plasma. In this study, the role of these ...lipids in TRALI was investigated using an isolated, perfused rat lung model. Rats were pretreated with endotoxin (LPS) or saline in vivo and the lungs were isolated, ventilated, and perfused with saline, or (a) 5% (vol/ vol) fresh human plasma, (b) plasma from stored blood from the day of isolation (D.0) or from the day of outdate (D.42), (c) lipid extracts from D.42 plasma, or (d) purified lysophosphatidylcholines. Lungs from saline or LPS-pretreated rats perfused with fresh (D.0) plasma showed no pulmonary damage as compared with saline perfused controls. LPS pretreatment/D.42 plasma perfusion caused acute lung injury (ALI) manifested by dramatic changes in both pulmonary artery pressure and edema. Incubation of LPS pre-tx rats with mibefradil, a Ca2+ channel blocker, or WEB 2170, a platelet-activating factor (PAF) receptor antagonist, inhibited ALI caused by D.42 plasma. Lung histology showed neutrophil sequestration without ALI with LPS pretreatment/saline or D.0 plasma perfusion, but ALI with LPS pretreatment/D.42 plasma perfusion, and inhibition of D.42 plasma induced ALI with WEB 2170 or mibefradil. A significant increase in leukotriene E4 was present in LPS-pretreated/D.42 plasma-perfused lungs that was inhibited by WEB 2170. Lastly, significant pulmonary edema was produced when lipid extracts of D.42 plasma or lysophosphatidylcholines were perfused into LPS-pretreated lungs. Lipids caused ALI without vasoconstriction, except at the highest dose employed. In conclusion, both plasma and lipids from stored blood produced pulmonary damage in a model of acute lung injury. TRALI, like the adult respiratory distress syndrome, may be the result of two insults: one derived from stored blood and the other from the clinical condition of the patient.
Platelet aggregation is a cardinal feature of both vascular repair and vascular disease. During aggregation platelets release a variety of vasoactive substances; some of these promote angiogenesis, ...endothelial permeability, and endothelial growth, actions shared by vascular endothelial growth factor (VEGF). This study was undertaken to investigate the hypothesis that VEGF is released by aggregating platelets. We found that VEGF was secreted during the in vitro aggregation of platelet-rich plasma induced by thrombin, collagen, epinephrine, and ADP (range 23-518 pg VEGF/ml). Furthermore, serum VEGF levels were elevated compared with plasma (230 +/- 63 vs. 38 +/- 8 pg VEGF/ml), indicative of VEGF release during whole blood coagulation. Lysates of apheresed, leukocyte-poor platelet units contained significant amounts of VEGF (2.4 +/- 0.8 pg VEGF/mg protein). VEGF message and protein were also present in a megakaryocytic cell line (Dami cell). These results suggest constitutive roles for platelet VEGF in the repair of intimal vessel injury and in the altered permeability and intimal proliferation seen at sites of platelet aggregation and thrombosis.
The cytokine Interferon-γ (IFN-γ) exerts powerful immunoregulatory effects on the adaptive immune system and also enhances functions of the neutrophil (PMN). The clinical use of IFN-γ has been driven ...by the finding that its administration to patients with chronic granulomatous disease (CGD) results in decreased incidence and severity of infections. However, IFN-γ has no effect on the characteristic defect of CGD, the inability to convert oxygen to microbicidal metabolites including superoxide anion (O2-) during the phagocytosis associated oxidative burst. We administered varying doses of IFN-γ to adult volunteers and studied the effects on plasma drug levels and response molecules and PMNs isolated from blood drawn at intervals over a 96- hour period. Plasma concentrations of IFN-γ, IP-10 and neopterin, and stimulated release of O2- from PMNs exhibited dose- and time-dependent increases after IFN-γ administration. Gene expression in PMNs was altered for 2775 genes; changes occurred rapidly after administration and returned to baseline in 24-36 hours. Several genes involved with neutrophil host defense were upregulated including those for components of the O2- generating NADPH oxidase; innate-immune and Fc receptors; proteins involved in MHCI and II; a regulator of circulating PMN number; guanylate binding proteins; and a key enzyme in synthesis of an essential NOS cofactor. Coordinate changes were detected in protein levels of representative products from several of these genes. Lysates from isolated neutrophils also demonstrated a spike in NO following IFN-γ administration. IFN-γ appears to increase non-oxygen dependent microbicidal functions of PMNs which could provide strategies to compensate for deficiencies, explain its clinical benefit for CGD patients and expand therapeutic applications of IFN-γ to other disorders. Trial registration: Protocol registered in ClinicalTrials.gov, NCT02609932, Effect of IFN-γ on Innate Immune Cells.
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