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Biomedical subjects

Christopher C Silliman

Publications and source records attributed to Christopher C Silliman.

At least 19 recordsLinked to original sources
PubMed2006

Donor antibodies to HNA-3a implicated in TRALI reactions prime neutrophils and cause PMN-mediated damage to human pulmonary microvascular endothelial cells in a two-event in vitro model.

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.

Blood Donors↗
PubMed2006

Soluble CD40 ligand accumulates in stored blood components, primes neutrophils through CD40, and is a potential cofactor in the development of transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a form of posttransfusion acute pulmonary insufficiency that has been linked to the infusion of biologic response modifiers (BRMs), including antileukocyte antibodies and lipids. Soluble CD40 ligand (sCD40L) is a platelet-derived proinflammatory mediator that accumulates during platelet storage. We hypothesized that human polymorpho-nuclear leukocytes (PMNs) express CD40, CD40 ligation rapidly primes PMNs, and sCD40L induces PMN-mediated cytotoxicity of human pulmonary microvascular endothelial cells (HMVECs). Levels of sCD40L were measured in blood components and in platelet concentrates (PCs) implicated in TRALI or control PCs that did not elicit a transfusion reaction. All blood components contained higher levels of sCD40L than fresh plasma, with apheresis PCs evidencing the highest concentration of sCD40L followed by PCs from whole blood, whole blood, and packed red blood cells (PRBCs). PCs implicated in TRALI reactions contained significantly higher sCD40L levels than control PCs. PMNs express functional CD40 on the plasma membrane, and recombinant sCD40L (10 ng/mL-1 mug/mL) rapidly (5 minutes) primed the PMN oxidase. Soluble CD40L promoted PMN-mediated cytotoxicity of HMVECs as the second event in a 2-event in vitro model of TRALI. We concluded that sCD40L, which accumulates during blood component storage, has the capacity to activate adherent PMNs, causing endothelial damage and possibly TRALI in predisposed patients.

CD40 Antigens↗
PubMed2006

Platelet-activating factor-induced clathrin-mediated endocytosis requires beta-arrestin-1 recruitment and activation of the p38 MAPK signalosome at the plasma membrane for actin bundle formation.

Clathrin-mediated endocytosis (CME) is a common pathway used by G protein-linked receptors to transduce extracellular signals. We hypothesize that platelet-activating factor (PAF) receptor (PAFR) ligation requires CME and causes engagement of beta-arrestin-1 and recruitment of a p38 MAPK signalosome that elicits distinct actin rearrangement at the receptor before endosomal scission. Polymorphonuclear neutrophils were stimulated with buffer or 2 microM PAF (1 min), and whole cell lysates or subcellular fractions were immunoprecipitated or slides prepared for colocalization and fluorescent resonance energy transfer analysis. In select experiments, beta-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with beta-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, beta-arrestin-1, and clathrin, 3) recruitment and activation of the apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK (ASK1/MKK3/p38 MAPK) signalosome, 4) cell polarization, and 5) distinct actin bundle formation at the PAFR. Neutralization of beta-arrestin-1 inhibited all of these cellular events, including PAFR internalization; conversely, dynamin-2 inhibition only affected receptor internalization. Selective p38 MAPK inhibition globally abrogated actin rearrangement; however, inhibition of MAPK-activated protein kinase-2 and its downstream kinase leukocyte-specific protein-1 inhibited only actin bundle formation and PAFR internalization. In addition, ASK1/MKK3/p38 MAPK signalosome assembly appears to occur in a novel manner such that the ASK1/p38 MAPK heterodimer is recruited to a beta-arrestin-1 bound MKK3. In polymorphonuclear neutrophils, leukocyte-specific protein-1 may play a role similar to fascin for actin bundle formation. We conclude that PAF signaling requires CME, beta-arrestin-1 recruitment of a p38 MAPK signalosome, and specific actin bundle formation at the PAFR for transduction before endosomal scission.

Actins↗
PubMed2006

The two-event model of transfusion-related acute lung injury.

The objective of this review is to present the two-event model of transfusion-related acute lung injury (TRALI), a life-threatening complication of transfusions that has been the most common cause of transfusion-related death over the past 2 yrs in the United States. The two-event model of TRALI, which is identical to the pathogenesis of the acute respiratory distress syndrome (ARDS), is reviewed and contrasted to antibody-mediated TRALI. Laboratory studies, both in vitro and in vivo, are discussed as well as human studies of TRALI. Methods to avoid patient exposure to blood components that may cause TRALI are also discussed.

Animals↗
PubMed2005

Transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a life-threatening adverse event of transfusion, which has an increasing incidence in the United States and is 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 transfusion of blood or blood components. A number of different models have been proposed to explain the pathogenesis. The first is an antibody-mediated event whereby transfusion of anti-HLA, class I or class II, or anti-granulocyte antibodies into patients whose leukocytes express the cognate antigens. The antibody:antigen interaction causes complement-mediated pulmonary sequestration and activation of neutrophils (PMNs) resulting in TRALI. The second is a two-event model: the first event is the clinical condition of the patient resulting in pulmonary endothelial activation and PMN sequestration, and the second event is the transfusion of a biologic response modifier (including anti-granulocyte antibodies, lipids, and CD40 ligand) that activates these adherent PMNs resulting in endothelial damage, capillary leak, and TRALI. These hypotheses are discussed with respect to animal models and human studies that provide the experimental and clinical relevance. The definition of TRALI, patient predisposition, treatment, prevention and reporting guidelines are also examined.

Animals↗
PubMed2005

Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation.

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is part of the microbicidal arsenal used by human polymorphonuclear neutrophils (PMNs) to eradicate invading pathogens. The production of a superoxide anion (O2-) into the phagolysosome is the precursor for the generation of more potent products, such as hydrogen peroxide and hypochlorite. However, this production of O2- is dependent on translocation of the oxidase subunits, including gp91phox, p22phox, p47phox, p67phox, p40phox, and Rac2 from the cytosol or specific granules to the plasma membrane. In response to an external stimuli, PMNs change from a resting, nonadhesive state to a primed, adherent phenotype, which allows for margination from the vasculature into the tissue and chemotaxis to the site of infection upon activation. Depending on the stimuli, primed PMNs display altered structural organization of the NADPH oxidase, in that there is phosphorylation of the oxidase subunits and/or translocation from the cytosol to the plasma or granular membrane, but there is not the complete assembly required for O2- generation. Activation of PMNs is the complete assembly of the membrane-linked and cytosolic NADPH oxidase components on a PMN membrane, the plasma or granular membrane. This review will discuss the individual components associated with the NADPH oxidase complex and the function of each of these units in each physiologic stage of the PMN: rested, primed, and activated.

Cell Membrane↗
PubMed2005

Reducing risk in infant cardiopulmonary bypass: the use of a miniaturized circuit and a crystalloid prime improves cardiopulmonary function and increases cerebral blood flow.

Advances in perfusion strategies have played an important role in improving outcomes following repair of complex congenital heart defects. The influence of cooling strategy, temperature, duration of circulatory arrest, and specific method of cerebral perfusion on neurologic morbidity have been extensively characterized. Similarly, the ability of pharmacologic agents to modulate the post-cardiopulmonary bypass (CPB) inflammatory response has been previously elucidated in both the laboratory and clinical arena. However, modification of the circuit and priming components have received comparably less attention. We recently showed that employment of a miniaturized circuit and a bloodless prime reduce inflammation and have salutary effects on cardiopulmonary function following hypothermic low-flow perfusion (HLF), and that this circuit may also improve cerebral protection following both deep hypothermic circulatory arrest and HLF. The current report, therefore, reviews current strategies utilized to minimize post-CPB inflammation and highlights the empirical evidence from our laboratory demonstrating the beneficial role of a miniaturized extracorporeal circuit in this context.

Animals↗
PubMed2005

Transfusion-related acute lung injury: definition and review.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is now the leading cause of transfusion-associated mortality, even though it is probably still underdiagnosed and underreported. NATIONAL HEART, LUNG, AND BLOOD INSTITUTE ACTION: 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. MAJOR CONCEPTS IN THE DEFINITION: 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. RECOMMENDATION: 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.

Blood Transfusion↗
PubMed2005

Clinically relevant osmolar stress inhibits priming-induced PMN NADPH oxidase subunit translocation.

BACKGROUND: The plasma membrane NADPH oxidase is responsible for the external generation of superoxide by neutrophils (polymorphonucleocytes [PMNs]). The oxidase is a multicomponent enzyme, active only when all subunits are translocated to and assembled at the membrane. We have recently demonstrated that platelet-activating factor (PAF) priming of PMNs translocates the cytosolic p67 subunit to the membrane position. Osmolar stress attenuates PAF priming of the oxidase. Consequently, we hypothesized that clinically relevant osmolar stress inhibits PAF priming-induced p67 translocation. METHODS: Isolated human PMNs were incubated at 37 degrees C for 5 minutes in buffer or 180 mmol/L hypertonic saline (HTS) followed by 3 minutes of incubation with or without 2 mumol/L PAF (resting, PAF, HTS, and HTS-PAF). Digital microscopy was used to determine p67 location in whole PMNs. Subcellular fractions were prepared and membrane translocation of p67 determined by protein electrophoresis. Resting cytosol fractions were immunodepleted of p67 and NADPH oxidase activity measured using p67-deficient sodium dodecyl sulfate cell-free oxidase assays: resting, PAF, or HTS-PAF membrane (1 mug) was combined with immunodepleted resting cytosol (25 mug). RESULTS: By all methodologies, PAF stimulated translocation of p67 to the PMN membrane and this translocation was prevented by osmolar stress (HTS-PAF). In cell-free oxidase assays, the membrane content of p67 after PAF stimulation was increased sufficiently to induce oxidase activity, whereas resting and HTS-PAF membrane did not (0.1 +/- 0.02, 0.23 +/- 0.04, and 0.14 +/- 0.04, respectively, p < 0.01) (resting versus HTS-PAF, no difference). CONCLUSION: PAF priming of the PMN oxidase involves translocation of p67 to the plasma membrane. Clinically relevant osmolar stress with hypertonic saline prevents this PAF-induced translocation of the p67 oxidase subunit. This finding provides new insight into the mechanisms responsible for osmolar control of PMN functional responses.

Cell Membrane↗
PubMed2004

Transfusion-related acute lung injury.

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.

Animals↗
PubMed2004

Transfusion of the injured patient: proceed with caution.

Transfusion of the injured patient with packed red blood cells (PRBCs) is a dynamic process requiring vigilance during the acute resuscitative and recovery phases postinjury. Although adverse events have been reported in 2% to 10% of injured patients, the advent of new detection techniques for viral pathogens has markedly decreased the risk of infectious transmission. However, transfusions are strongly associated with immunosuppression in the host, which may occur days after the initial injury and may lead to bacterial infections. Conversely, early transfusion of stored PRBCs, > 6 units in the first 12 h postinjury, contributes to an early state of hyperinflammation that is a strong, independent predictor of multiple organ failure (MOF) in those patients with intermediate injury severity scores. The roles of prestorage leukoreduction are also reviewed with respect to the promotion of both immunosuppression and hyperinflammation. We further summarize studies with hemoglobin substitutes, whose use may obviate many of the untoward events of transfusion and promise to lead to better outcomes for injured patients.

Erythrocyte Transfusion↗
PubMed2004

Transfusion-induced leukocyte IL-8 gene expression is avoided by the use of human polymerized hemoglobin.

BACKGROUND: Red blood cell (pRBC) transfusion is an independent risk factor for multiple organ failure (MOF); a maladaptive immuno-inflammatory response is implicated. Interleukin-8 (IL-8) is one putative mediator of this response. We previously observed that injured patients resuscitated with pRBCs have increased plasma IL-8 compared with those given human polymerized hemoglobin (PolyHb). To further elucidate the mechanisms responsible for this difference in IL-8, we devised an ex-vivo transfusion model. We hypothesize that pRBC transfusion induces increased IL-8 gene expression that is avoided by the use of PolyHb. METHODS: Human volunteer blood was incubated alone (RB) or with a major transfusion (50% exchange) of either post-storage leukoreduced O-pRBCs (RB + pRBC) or PolyHb (RB + PolyHb) for 30 minutes at 37 degrees C. Total leukocyte (TL) or polymorphonuclear leukocyte (PMN) total RNA was isolated and IL-8 mRNA quantified. Results are reported as amol IL-8 mRNA/microg total RNA +/- SEM. Stats: ANOVA with Bonferroni/Dunn post hoc analysis. RESULTS: Simulated transfusion of pRBCs increased TL IL-8 mRNA (RB=0.28 +/- 0.10 amol/microg total RNA, RB + pRBC=2.24 +/- 0.25 amol/microg total RNA, p <0.01), whereas PolyHb did not (B + PolyHb=0.82 +/- 0.30 amol/microg total RNA). PolyHb IL-8 mRNA was less than pRBC transfused (p <0.01). In PMNs, simulated transfusion of pRBCs increase IL-8 mRNA (RB=3.17 +/- 1.05 amol/microg total RNA, RB + pRBC=7.60 +/- 1.79 amol/microg total RNA, p <0.01), whereas PolyHb did not (RB + PolyHb=4.53 +/- 1.64 amol/microg total RNA). CONCLUSIONS: Stored pRBCs induces increased TL and PMN IL-8 gene expression, whereas human polymerized hemoglobin, in lieu or pRBCs, avoids this increase. These experimental results corroborate our previous clinical studies and further encourage the study of PolyHb as a resuscitation strategy to decrease postinjury MOF.

Analysis of Variance↗
PubMed2004

Systemic neutrophil priming by lipid mediators in post-shock mesenteric lymph exists across species.

BACKGROUND: Post-hemorrhagic shock mesenteric lymph (PHSML) has been linked with neutrophil (PMN) priming, endothelial cell (EC) activation, and acute lung injury (ALI) in rodent models. We have previously identified the lipid fraction of PHSML as containing the causative agent(s). Due to the lesson learned from the rodent gut bacterial translocation experience, we sought to confirm this phenomenon using a large animal model; hypothesizing that lymph collected from the porcine gut following ischemia/reperfusion (I/R) would cause PMN priming. METHODS: Mesenteric lymph was collected from adult pigs before, during, and for 2 hours after non-lethal hemorrhagic shock (mean arterial pressure = 30 mm Hg x 45 minutes). Whole lymph and the extracted lipid fractions of the lymph were then added to isolated human and porcine PMNs and superoxide production was measured by cytochrome C reduction. RESULTS: Hemorrhagic shock profoundly affected mesenteric lymph flow from baseline (pre-shock) flow rates of 75.63 +/- 8.86 mL/hr to 49.38 +/- 5.76 mL/hr during shock and increasing to 253.38 +/- 27.62 mL/hr after 2 hours of resuscitation. Human PMNs exposed to both whole lymph (PHSML) and its extracted lipids (PHSML Lipid) collected 2 hours after shock exhibited more than a two-fold increase in superoxide release upon activation compared with pre-shock samples: PHSML- 6.27 +/- 0.83 versus 2.56 +/- 0.60 nmolO2(-)/ 3.75 cells/mL/min, respectively (p = 0.007), PHSML Lipid- 4.93 +/- 0.34 versus 2.49 +/- 0.11 nmolO2(-)/ 3.75 cells/mL/min (p < 0.001). Similarly, porcine PMNs exhibited close to a two-fold activation when exposed to the lymph and lipid fraction: PHSML- 4.51 +/- 0.42 versus 1.06 +/- 0.28 nmolO2(-)/ 3.75 cells/mL/min (p = 0.008), PHSML Lipid-4.80 +/- 0.81 versus 1.55 +/- 0.23 nmolO2(-)/ 3.75 cells/mL/min (p = 0.002). CONCLUSION: Mesenteric lymphatics serve as the conduit for inflammatory mediators elaborated by the post-ischemic gut in both small and large animal models. Further, the causal agent(s) exist in the lipid fraction of the lymph and are active on both human and animal PMNs.

Animals↗
PubMed2004

Joint ASH and AABB educational session.

In the vein-to-vein flow of blood from donor to patient, the role of the transfusion medicine specialist has become increasingly centered at the bedside. Three clinically centered issues in blood safety and in blood conservation are presented in this chapter. In Section I, Dr. Patricia Hewitt presents the epidemiologic and clinical evidence regarding new variant Creutzfeldt-Jakob disease (nvCJD) in the UK and its relevance to transfusion medicine. Lessons learned from the responses by the National Blood Service to this crisis are discussed, particularly in the context of recent evidence of a case of vCJD transmission by blood transfusion and a second case of apparent transmission of abnormal prion protein without development of clinical illness. In Section II, Dr. Christopher Silliman and his colleagues summarize recent knowledge gained regarding transfusion-related acute lung injury (TRALI), which is now the leading cause of transfusion-related mortality. Two different etiologies have been proposed: a single antibody-medicated event, involving anti-HLA Class I and Class II, or anti-granulocyte antibodies; and a two-event model, which includes the clinical condition of the patient resulting in pulmonary endothelial activation and neutrophil sequestration. The second event is the transfusion of a biologic response modifier (lipids or antibodies) in the blood component that activates primed neutrophils. Prevention, clinical treatment, and proposed definition of TRALI are discussed. In Section III, Dr. Lawrence Goodnough and colleagues present a transfusion medicine service approach to the utilization of recombinant factor VIIa (rFVIIa) in non-approved clinical settings. rFVIIa has a potential role as a hemostatic intervention in a variety of clinical settings, yet few clinical trials have been completed to date to guide indications for its use. The policies presented here are those in place at the authors' medical center, and will undergo periodic review and revision as relevant new information and data are generated.

Blood Transfusion↗
PubMed2003

Formyl-Met-Leu-Phe induces calcium-dependent tyrosine phosphorylation of Rel-1 in neutrophils.

Chemoattractant priming and activation of PMNs results in changes in cytosolic Ca2+ concentration, tyrosine kinase activity, and gene expression. We hypothesize that the initial signaling for the activation of a 105kDa protein (Rel-1) requires Ca2+-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 30s. 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 Ca2+ but chelation negated the effects, including the cytosolic Ca2+ flux, oxidase activation, and the tyrosine phosphorylation of Rel-1. Conversely, chelation of extracellular Ca2+ decreased the fMLP-mediated Ca2+ 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 Ca2+. Thus, fMLP concentrations that prime or activate the oxidase cause a rapid Ca2+-dependent tyrosine phosphorylation of Rel-1 involving p38 MAPK activation.

Calcium↗
PubMed2003

Plasma and lipids from stored platelets cause acute lung injury in an animal model.

BACKGROUND: Transfusion of PLT concentrates may cause TRALI, a life-threatening reaction that has been linked to the infusion of anti-WBC immunoglobulins or older, stored PLTs that contain bioactive lipids. We hypothesize that lipids generated during storage of PLTs cause TRALI in a two-event animal model. STUDY DESIGN AND METHODS: Plasma from both whole-blood PLTs (WB-PLTs) and apheresis PLTs (A-PLTs) was isolated on Day 0 (D.0) and Day 5 (D.5) of storage and heat-treated before use. Rats were pretreated with saline or 2 mg per kg endotoxin (LPS), anesthetized, and the lungs were ventilated, isolated, and perfused with saline or 5-percent PLT plasma. Pulmonary artery pressure, pulmonary edema, and leukotriene B4 levels (perfusate) were measured. RESULTS: Plasma from D.5, but not D.0, of the identical WB-PLT and A-PLT units caused injury in lungs from LPS-pretreated rats (LPS/D.5) evidenced by increases in pulmonary edema and leukotriene B4 (p < 0.05). Lipid extracts and purified lipids from D.5 PLT plasma also elicited injury in lungs from LPS-pretreated rats (p < 0.05). Saline/D.5 plasma or lipids or LPS/D.0 did not cause pulmonary edema. Prestorage WBC reduction was ineffective in inhibiting TRALI. CONCLUSION: PLT-induced TRALI may be the result of two events: 1) the clinical condition of the patient and 2) the infusion of lipids in stored PLTs.

Acute Disease↗