RESUMEN
BACKGROUND: The Zika virus (ZIKV) epidemic of 2015/2016 spread throughout numerous countries. It emerged in mainland Latin America and spread to neighboring islands, including the Caribbean island of Barbados. Recent studies have indicated that the virus must have already been circulating in local mosquito populations in Brazil for almost 2 years before it was identified by the World Health Organization in 2015. Metagenomic detection assays have the potential to detect emerging pathogens without prior knowledge of their genomic nucleic acid sequence. Yet their applicability as vector surveillance tools has been widely limited by the complexity of DNA populations from field-collected mosquito preparations. The aim of this study was to investigate local vector biology and characterize metagenomic arbovirus diversity in Aedes mosquitoes during the ongoing 2015/2016 ZIKV epidemic. METHODS: We performed a short-term vector screening study on the island of Barbados during the ongoing 2015/2016 ZIKV epidemic, where we sampled local Aedes mosquitoes. We reanalyzed mosquito viral microbiome data derived from standard Illumina MiSeq sequencing to detect arbovirus sequences. Additionally, we employed deep sequencing techniques (Illumina HiSeq) and designed a novel bait capture enrichment assay to increase sequencing efficiency for arbovirus sequences from complex DNA samples. RESULTS: We found that Aedes aegypti seemed to be the most likely vector of ZIKV, although it prevailed at a low density during the observed time period. The number of detected viruses increased with sequencing depth. Arbovirus sequence enrichment of metagenomic DNA preparations allowed the detection of arbovirus sequences of two different ZIKV genotypes, including a novel one. To our knowledge, this is the first report of the S3116W mutation in the NS5 gene region of ZIKV polyprotein. CONCLUSIONS: The metagenomic arbovirus detection approach presented here may serve as a useful tool for the identification of epidemic-causing arboviruses with the additional benefit of enabling the collection of phylogenetic information on the source. Apart from detecting more than 88 viruses using this approach, we also found evidence of novel ZIKV variants circulating in the local mosquito population during the observed time period.
Asunto(s)
Aedes/virología , Variación Genética , Metagenómica , Virus Zika/genética , Animales , Barbados , Epidemias/estadística & datos numéricos , Mosquitos Vectores/virología , Filogenia , Virus Zika/clasificación , Infección por el Virus Zika/transmisiónRESUMEN
BACKGROUND: The causes of oedematous vs non-oedematous childhood malnutrition (OM vs NOM) remain elusive. It is possible that inherited differences in handling oxidant stressors are a contributing factor. AIMS: To test for associations between polymorphisms in five genes and (i) risk of OM, a case-control study, and (ii) percentage cytotoxicity in peripheral blood mononuclear cells (PBMCs) exposed to hydrogen peroxide (H(2)O(2)), an in vitro cell challenge study. METHODS: Participants had been admitted previously for treatment of OM (cases, n = 74) or NOM (controls, n = 50), or were an independent set of healthy pregnant women (n = 47) who donated peripheral blood mononuclear cells. We tested for associations between genetic variation and outcome using single markers or a bivariate score constructed by counting numbers of deleterious alleles for each of 15 possible pairs of markers. RESULTS: In the case-control study there were no significant single-marker associations with OM. We did find that higher bivariate scores were associated with OM for the pair of NAD(P)H:quinone oxidoreductase 1 and catalase (odds ratio 2·00, 95% CI 1·05-3·82). In the cell challenge experiments, there were no significant associations with percentage cytotoxicity. CONCLUSIONS: Variation in this small set of genes seems unlikely to have a large impact on either risk of OM or cytotoxicity after H(2)O(2) exposure. The use of larger sample sizes to test the effects of a much larger set of genetic variants will be required in order to determine whether genetic variation contributes to the risk of OM. Such studies have potential for improving our understanding of causal pathways in OM.
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Trastornos de la Nutrición del Niño/enzimología , Trastornos de la Nutrición del Niño/genética , Leucocitos Mononucleares/enzimología , Estrés Oxidativo , Estudios de Casos y Controles , Niño , Preescolar , Edema/genética , Edema/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Lactante , Leucocitos Mononucleares/metabolismo , EmbarazoAsunto(s)
Factor de Necrosis Tumoral alfa/metabolismo , Cicatrización de Heridas/fisiología , Animales , Apoptosis/genética , Apoptosis/fisiología , Diabetes Mellitus Experimental/fisiopatología , Modelos Animales de Enfermedad , Humanos , Inflamación/genética , Inflamación/fisiopatología , Ratones , FN-kappa B/genética , FN-kappa B/fisiología , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
OBJECTIVE: Extravascular trafficking of leukocytes into organs is thought to play a major role in the pathophysiologic mechanisms of the inflammatory response to cardiopulmonary bypass, yet leukocyte extravasation is difficult to study clinically. Here we have tested the hypothesis that leukocyte emigration into skin blisters can provide a way to monitor the inflammatory effect of cardiopulmonary bypass that allows testing of anti-inflammatory interventions (exemplified by aprotinin). METHODS: Patients undergoing primary elective coronary artery bypass grafting (n = 14) were randomized into 2 equal groups to receive saline infusion during cardiopulmonary bypass (control group) or high-dose aprotinin. Experimental skin blisters (in duplicate) were induced on the forearm by means of topical application of the vesicant cantharidin, and blister fluid was sampled at 5 hours postoperatively. Inflammatory leukocyte subsets in blister fluid were analyzed by means of flow cytometry by using expression of CD11b and CD62L as a phenotypic marker of activation. RESULTS: In the control group of patients, cardiopulmonary bypass surgery triggered a 381% increase in leukocyte extravasation into the skin compared with reference blisters carried out before surgical intervention, with neutrophil (P = .014), monocyte (P = .014), and eosinophil (P = .009) levels all statistically significantly increased. In the aprotinin group there was no statistically significant increase during cardiopulmonary bypass surgery in any inflammatory leukocyte subset. The activation phenotype of extravascular leukocytes was not significantly altered between surgical groups. CONCLUSIONS: This study introduces the cantharidin blister technique as a powerful new research tool for analyzing the inflammatory effect of cardiopulmonary bypass in vivo. It has provided detailed molecular insight into the extravascular leukocyte population during cardiopulmonary bypass. Although aprotinin blocked cardiopulmonary bypass-dependent extravasation of leukocytes, there was no change in their CD11b/CD62L activation status. The cantharidin skin test thus represents a novel research tool for evaluating future anti-inflammatory interventions in cardiothoracic surgery.
Asunto(s)
Puente Cardiopulmonar/efectos adversos , Inflamación/inmunología , Leucocitos/inmunología , Anciano , Vesícula/inmunología , Puente de Arteria Coronaria , Femenino , Humanos , Inflamación/fisiopatología , Masculino , Persona de Mediana EdadRESUMEN
Cardiopulmonary bypass, although remaining an indispensable asset in cardiac surgery, especially in more complex and repeat operations, is associated with significant thrombin generation in the bypass circuit, leading to the activation of platelets, the coagulation system, an inflammatory response, and perioperative stroke. Recent clinical studies and meta-analyses of clinical trials in coronary artery bypass grafting surgery have confirmed that aprotinin not only reduces transfusion requirements in cardiac surgery but also confers significant protection against platelet dysfunction, activation of the systemic inflammatory response, and perioperative stroke when administered at the full (or "Hammersmith") dose. This article reviews research from several independent groups to propose a novel mechanism through which the antithrombotic, anti-inflammatory, and neuroprotective mechanism might be mediated, via protection of the high-affinity thrombin receptor protease-activated receptor 1 (PAR1).
Asunto(s)
Aprotinina/farmacología , Puente Cardiopulmonar/efectos adversos , Receptor PAR-1/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/farmacología , Accidente Cerebrovascular/prevención & control , Síndrome de Respuesta Inflamatoria Sistémica/prevención & control , Trombosis/prevención & control , Animales , Aprotinina/uso terapéutico , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Puente Cardiopulmonar/mortalidad , Ensayos Clínicos como Asunto , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Receptor PAR-1/metabolismo , Inhibidores de Serina Proteinasa/uso terapéutico , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Trombina/metabolismo , Trombosis/etiología , Trombosis/metabolismoRESUMEN
BACKGROUND: Protease-activated receptor-1 (PAR1) is the principal thrombin receptor in the vasculature, and antagonists against this receptor are in preclinical trials. Aprotinin, already approved for clinical use to reduce transfusion requirements in cardiopulmonary bypass (CPB) surgery, has been shown to inhibit PAR1 activation in vitro. Here, we exploit CPB as a model for thrombin generation in humans to examine whether aprotinin can inhibit platelet PAR1 activation clinically. METHODS AND RESULTS: PAR1 expression and function on platelets was examined in coronary artery bypass grafting (CABG) patients randomized into 2 groups: (1) those receiving saline infusion during CPB (n=17) and (2) those receiving aprotinin (2x10(6) kallikrein inhibitor units [KIU] in pump prime, 2x10(6) KIU loading dose, followed by 0.5x10(6) KIU/h [n=13]). Platelets in the saline group showed loss of PAR1-specific function at 2 hours after CPB, but this was preserved in the aprotinin group (P<0.001). These effects were most likely targeted at PAR1 receptor cleavage, because (1) the level of thrombin generated during CPB did not vary significantly between groups, (2) expression of SPAN12, which detects only uncleaved PAR1 receptors, was preserved in the aprotinin but not the placebo group (P<0.05), and (3) supporting evidence in vitro showed reduced thrombin-induced PAR1 cleavage (P<0.001) and platelet aggregation (P<0.001) in the presence of aprotinin. CONCLUSIONS: This study demonstrates that platelet PAR1 activation by thrombin can be inhibited by aprotinin. Our results extend the clinical mechanism of action of aprotinin and provide the first proof of principle that PAR1 can be inhibited clinically. This has implications beyond cardiac surgery for the development of therapeutic PAR1 blockade.
Asunto(s)
Aprotinina/uso terapéutico , Puente Cardiopulmonar , Receptor PAR-1/antagonistas & inhibidores , Humanos , Trombina/metabolismoRESUMEN
OBJECTIVE: To study the effect of unfractionated heparin (UFH) versus low molecular weight heparin (LMWH) in combination with glycoprotein (Gp) IIb/IIIa blockers on platelet activation and aggregation. METHODS: Washed platelets were stimulated with thrombin in the presence or absence of UFH (monoparin), LMWH (enoxaparin), and a Gp IIb/IIIa blocker (abciximab, eptifibatide, or tirofiban). RESULTS: Although Gp IIb/IIIa antagonists blocked the final common pathway of thrombin induced platelet aggregation, UFH and LMWH were better at blocking upstream platelet activation. UFH was significantly more effective than LMWH at inhibiting P selectin expression (p = 0.001) and platelet derived growth factor release from thrombin activated platelets (p = 0.012). CONCLUSIONS: UFH and LMWH exert complementary effects to Gp IIb/IIIa blockers by inhibiting afferent pathways of platelet activation. Coadministration of heparin with Gp IIb/IIIa blockers provides improved protection against persistent platelet activation, thereby improving outcome after percutaneous coronary intervention. Judging from these data, UFH may be more effective in this regard than LMWH, at least in vitro. The use of LMWH in preference to UFH during percutaneous coronary intervention, although initially attractive, may inadequately protect against platelet activation despite the presence of Gp IIb/IIIa blockers.
Asunto(s)
Anticoagulantes/farmacología , Hemostáticos/farmacología , Heparina/farmacología , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Trombina/farmacología , Tirosina/análogos & derivados , Abciximab , Angioplastia Coronaria con Balón , Anticuerpos Monoclonales/farmacología , Reestenosis Coronaria/sangre , Enoxaparina/administración & dosificación , Enoxaparina/farmacología , Eptifibatida , Citometría de Flujo , Heparina/administración & dosificación , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Péptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/administración & dosificación , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Tirofibán , Tirosina/farmacologíaRESUMEN
The recently described hemoglobin scavenger receptor CD163 mediates the endocytosis of hemoglobin:haptoglobin (Hb:Hp) complexes and thereby counters Hb-induced oxidative tissue damage after hemolysis. Although CD163 has been indirectly associated with antiinflammatory and atheroprotective activity, no ligand-receptor-effector pathway has yet been described for this receptor. To understand the significance of CD163 and more clearly define downstream pathways linked to inflammatory resolution, we studied the expression and function of CD163 in human monocytes/macrophages using both in vitro and in vivo models. Differentiation of human blood monocytes into macrophages either by in vitro culture or in resolving cantharidin-induced skin blisters led to an equivalent increase (>15x) in CD163 expression. Elevated CD163 levels were also noted on circulating monocytes in cardiac surgical patients during the resolution phase of the systemic inflammatory response to cardiopulmonary bypass surgery. In each case, binding of Hb:Hp to CD163-bearing cells elicited potent interleukin-10 secretion, and this was inhibited by the anti-CD163 antibody RM3/1. Release of interleukin-10, in turn, induced heme oxygenase-1 stress protein synthesis via an autocrine mechanism. Such induction of heme oxygenase-1 was observed in vivo 24 to 48 hours after the onset of cardiopulmonary bypass surgery. These results identify novel antiinflammatory and cytoprotective effector pathways in human monocytes/macrophages related to Hb scavenging and metabolism, which may have relevance in atheroprotection, wound healing, and patient recovery postoperatively.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/fisiología , Puente Cardiopulmonar , Hemo Oxigenasa (Desciclizante)/biosíntesis , Interleucina-10/biosíntesis , Macrófagos/inmunología , Monocitos/inmunología , Receptores de Superficie Celular/fisiología , Anciano , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Comunicación Autocrina , Vesícula/inmunología , Células Cultivadas , Puente de Arteria Coronaria , Femenino , Haptoglobinas/metabolismo , Hemo-Oxigenasa 1 , Hemoglobinas/metabolismo , Humanos , Inflamación/enzimología , Inflamación/metabolismo , Macrófagos/enzimología , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Monocitos/enzimología , Receptores de Superficie Celular/metabolismoRESUMEN
The clinical benefit of aprotinin with respect to improved hemostasis, platelet function, and inflammatory response to cardiopulmonary bypass (CPB) surgery has been well documented, but these benefits have been overshadowed by the concern that such a potently hemostatic agent might also be prothrombotic. In this article, we discuss recent advances in the understanding of the basic mechanism of aprotinin that have led to the identification of new antiinflammatory targets and the discovery that aprotinin is, in fact, antithrombotic with respect to platelets. Its antithrombotic action is mediated by the selective blocking of the major thrombin receptor, the protease-activated receptor 1 (PAR1), but not other receptors of platelet activation (ie, collagen, adenosine diphosphate [ADP], or epinephrine receptors). The selective targeting of PAR1 enables aprotinin to protect platelets from unwanted activation by thrombin generated during CPB surgery (consistent with a role in platelet-preservation), while permitting the participation of platelets in the formation of hemostatic plugs at wound and suture sites, where collagen, ADP, and epinephrine are most likely to be expressed. Aprotinin therefore exerts a subtle hemostatic yet antithrombotic mechanism of action, which, when allied with its multitiered antiinflammatory effect, makes this drug a valuable companion to cardiac surgery.
Asunto(s)
Antiinflamatorios/farmacología , Aprotinina/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Plaquetas/efectos de los fármacos , Puente Cardiopulmonar , Adhesión Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Humanos , Leucocitos/efectos de los fármacos , Receptor PAR-1 , Receptores de Trombina/antagonistas & inhibidoresRESUMEN
Controversy continues as to whether aprotinin (Trasylol) is prothrombotic. The recent discovery of the thrombin receptor family, known as the protease-activated receptor family (PAR) has been essential in aiding our understanding of the mechanism of action of aprotinin. Our results show that aprotinin has no effect on platelet aggregation induced by adrenaline, adenosine diphosphate, phorbol-12-myristate-13-acetate, collagen or PAR 1 agonist peptide. However, aprotinin inhibits thrombin-induced platelet activation as assessed by macroaggregation, microaggregation and platelet membrane calcium flux. Aprotinin inhibits proteolytic activation of platelets, but platelets can still be activated by non-proteolytic mechanisms.
Asunto(s)
Aprotinina/efectos adversos , Hemostáticos/efectos adversos , Inhibidores de Proteasas/efectos adversos , Receptores de Trombina/efectos de los fármacos , Trombofilia/inducido químicamente , Adenosina Difosfato/farmacología , Aprotinina/farmacología , Señalización del Calcio/efectos de los fármacos , Colágeno/farmacología , Epinefrina/farmacología , Proteínas de Unión al GTP/fisiología , Hemostáticos/farmacología , Humanos , Modelos Biológicos , Modelos Moleculares , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Receptor PAR-1 , Receptores de Trombina/química , Acetato de Tetradecanoilforbol/farmacología , Trombina/farmacología , Tripsina/farmacologíaRESUMEN
BACKGROUND: Cardiopulmonary bypass surgery is often accompanied by a systemic inflammatory response, which can lead to postoperative complications in high-risk patients. This is mediated in part through a systemic rise in inflammatory cytokine levels and the sequestration of leukocytes within organs. Aprotinin has previously been shown to exert an anti-inflammatory effect by preventing the capacity of leukocytes to transmigrate through vascular endothelium. Here we have focused on whether aprotinin has an effect on endothelial cell activation and adhesion molecule expression in response to tumor necrosis factor-alpha, particularly with reference to whether aprotinin inhibits tumor necrosis factor-stimulated neutrophil transendothelial migration. METHODS AND RESULTS: Intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expression was studied in tumor necrosis factor-alpha-activated human umbilical vein endothelial cells in the presence of aprotinin at 200, 800, and 1600 kIU/mL. Aprotinin inhibited tumor necrosis factor-alpha-stimulated expression of intercellular adhesion molecule-1 (P =.019 at 1600 kIU/mL) and vascular cell adhesion molecule-1 (P =.003 at 1600 kIU/mL) but not E-selectin. Similar results were obtained in the dermal microvascular endothelial cell line, HMEC-1, which exhibited diminished intercellular adhesion molecule-1 expression in the presence of aprotinin (P =.040 at 800 kIU/mL and P <.001 at 1600 kIU/mL). Aprotinin also significantly inhibited neutrophil transmigration across tumor necrosis factor-alpha-activated human umbilical vein endothelial cells (P =.046 at 1600 kIU/mL). CONCLUSIONS: We have demonstrated that aprotinin inhibits intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, but not E-selectin, expression on tumor necrosis factor-alpha-activated endothelial cells and that transendothelial migration by neutrophils is also specifically suppressed under these conditions. Our results indicate that endothelial cells can be specifically targeted by aprotinin, therefore adding to our understanding of the anti-inflammatory mechanism of action of aprotinin during cardiopulmonary bypass.
Asunto(s)
Aprotinina/farmacología , Endotelio Vascular/efectos de los fármacos , Inhibidores de Serina Proteinasa/farmacología , Factor de Necrosis Tumoral alfa/fisiología , Puente Cardiopulmonar , Moléculas de Adhesión Celular/efectos de los fármacos , Células Cultivadas , Selectina E/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Citometría de Flujo , Humanos , Mediadores de Inflamación/fisiología , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacosRESUMEN
Crystals are an important cause of inflammatory rheumatic diseases and provide relatively simple paradigms for modelling inflammatory responses in general. Thus, in the case of gout, we know that hyperuricemia leads to precipitation of monosodium urate (MSU) crystals in joints, which are taken up by leukocytes, and then an acute attack of arthritis is triggered. However, fundamental questions remain unanswered. Why are only certain hyperuricemic individuals, and then only certain joints, affected? What factors maintain joints in a quiescent state, what prompts the resolution of an inflammatory attack, and are these related? This article draws on developments during the past year to support the idea that the mononuclear phagocyte may play a key role within the synovial compartment, tipping the balance from the asymptomatic state to acute inflammation, or vice versa, depending on their state of monocyte to macrophage differentiation.
Asunto(s)
Artritis/inducido químicamente , Artritis/metabolismo , Gota/inducido químicamente , Gota/metabolismo , Cristalización , Humanos , Macrófagos/fisiología , Monocitos/fisiología , Neutrófilos/fisiología , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Ácido Úrico/efectos adversos , Ácido Úrico/sangreRESUMEN
Before the discovery of its hemostatic properties, aprotinin was thought of as a potential anti-inflammatory agent. Its clinical introduction in 1987 to prevent blood loss during cardiac surgery [Royston 1987, van Oeveren 1987] led to its anti-inflammatory benefits being largely overlooked in favor of a vigorous debate centering on whether aprotinin may be pro-thrombotic when given to patients. In this article, we summarize evidence for the anti-inflammatory activity of aprotinin and discuss our recent contributions in this area. We also summarize the state of the thrombosis debate and discuss our recent evidence from purified platelets which shows that aprotinin is simultaneously hemostatic yet anti-thrombotic.
Asunto(s)
Aprotinina/uso terapéutico , Puente Cardiopulmonar/efectos adversos , Hemostáticos/uso terapéutico , Embolia Intracraneal/prevención & control , Síndrome de Respuesta Inflamatoria Sistémica/prevención & control , Animales , Aprotinina/farmacología , Hemostáticos/farmacología , Humanos , Inflamación/prevención & control , Embolia Intracraneal/etiología , Leucocitos/efectos de los fármacos , Síndrome de Respuesta Inflamatoria Sistémica/etiologíaRESUMEN
Aprotinin (Trasylol) is generally regarded to be an effective hemostatic agent that prevents blood loss and preserves platelet function during cardiac surgery procedures requiring cardiopulmonary bypass (CBP). However, its clinical use has been limited by the concern that such a potent hemostatic agent might be prothrombotic, particularly in relation to coronary vein graft occlusion. In this review we present a mechanism of action that challenges such a viewpoint and explains how aprotinin can be simultaneously hemostatic and antithrombotic. Aprotinin achieves these two apparently disparate properties by selectively blocking the proteolytically activated thrombin receptor on platelets, the protease-activated receptor 1 (PAR1), while leaving other mechanisms of platelet aggregation unaffected. We also review recent research leading to the discovery of novel antiinflammatory targets for aprotinin. A better understanding of its mechanisms of action has led to the conclusion that aprotinin is a remarkable drug with the capacity to correct many of the imbalances that develop in the coagulation system and the inflammatory system after CPB. Nonetheless, it has been clinically underused for fear of causing thrombotic complications, a fear that in light of recent evidence may be unfounded.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Aprotinina/farmacología , Puente Cardiopulmonar , Fibrinolíticos/farmacología , Hemostáticos/farmacología , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Aprotinina/efectos adversos , Puente de Arteria Coronaria , Fibrinolíticos/efectos adversos , Oclusión de Injerto Vascular/sangre , Oclusión de Injerto Vascular/prevención & control , Hemostáticos/efectos adversos , HumanosRESUMEN
The cardiopulmonary bypass (CPB)-related inflammatory response involves leucocyte activation and increased leucocyte-endothelial cell interaction. L-selectin is an adhesion molecule expressed on the surface of leucocytes which participates in the initial rolling step of the leucocyte-endothelial cell adhesion cascade. L-selectin is proteolytically cleaved off the surface of leucocytes when they become activated, an event that is regarded as a marker of leucocyte activation. Aprotinin is a protease inhibitor that has been used in cardiac surgery as a haemostatic agent and also exhibits certain anti-inflammatory properties. In this study, peripheral venous blood from volunteers was pre-incubated with aprotinin at 200, 800 and 1600 kallikrein inhibiting units (kiu)/ml and stimulated with the chemoattractants N-formyl-methyl-leucyl-phenylalanine (fMLP) or platelet activating factor (PAF). Surface expression of L-selectin on neutrophils was measured using a monoclonal antibody and flow cytometry. The results demonstrate that aprotinin inhibits shedding of L-selectin in a dose-dependent fashion (p=0.0278 and 0.0005, respectively, at 800 and 1600 kiu/ml for fMLP-stimulated shedding; p=0.0017 and 0.0010, respectively, at 200 and 800 kiu/ml for PAF-stimulated shedding). This effect may be of significance with respect to the anti-inflammatory action of aprotinin in patients undergoing CPB.
Asunto(s)
Selectina L/efectos de los fármacos , Selectina L/metabolismo , Neutrófilos/metabolismo , Aprotinina/farmacología , Puente Cardiopulmonar/efectos adversos , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Inflamación/prevención & control , N-Formilmetionina Leucil-Fenilalanina/farmacología , Activación Neutrófila/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
BACKGROUND: The transplantation of pig organs into humans requires a detailed knowledge of similarities and differences between the two species in the molecular physiology of host defense mechanisms. We therefore set out to identify porcine intercellular adhesion molecule (ICAM)-1 and to characterize its expression by endothelial cells. METHODS: Porcine ICAM-1 cDNA was isolated from an endothelial cell cDNA library. An anti-pig ICAM-1 monoclonal antibody was generated and used to investigate the regulation by cytokines of ICAM-1 expression by porcine aortic endothelial cells (PAEC), using flow cytometry. RESULTS: We found that porcine ICAM-1 was similar in primary structure to human ICAM-1, with five Ig-like domains. COS-7 cells transfected with porcine ICAM-1 supported beta2 but not alpha4 integrin-dependent adhesion of human T lymphoblasts. There was a low-level surface expression of ICAM-1 on unstimulated PAEC and increased expression after stimulation with tumor necrosis factor (TNF)-alpha. However expression of ICAM-1 seemed to be significantly lower than that of vascular cell adhesion molecule-1, both on unstimulated and TNF-alpha-activated PAEC. Recombinant porcine interferon-gamma weakly stimulated ICAM-1 expression when incubated alone with PAEC but had an inhibitory effect on the increase in ICAM-1 due to TNF-alpha, both at 8 and 24 hr. CONCLUSIONS: Our observations confirm the existence of ICAM-1 in the pig and provide novel insights into how porcine and human endothelial cells differ in terms of adhesion molecule expression and cytokine responsiveness. Such differences are potentially important in interpreting models of inflammation in the pig and also in understanding the process of rejection of porcine xenografts.
Asunto(s)
Citocinas/farmacología , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/genética , Secuencia de Aminoácidos , Animales , Células COS , Adhesión Celular , Endotelio Vascular/efectos de los fármacos , Biblioteca de Genes , Humanos , Molécula 1 de Adhesión Intercelular/química , Molécula 1 de Adhesión Intercelular/fisiología , Interferón gamma/farmacología , Interleucinas/farmacología , Cinética , Linfocitos/fisiología , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Porcinos , Transcripción Genética , Transfección , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVE: We have hypothesized that the process of monocyte to macrophage differentiation may alter the inflammatory response of mononuclear phagocytes to the uptake of monosodium urate monohydrate (MSU) crystals. METHODS: Eight mouse monocyte/macrophage cell lines were arranged in increasing order of differentiation, as judged by expression of the macrophage markers F4/80 and BM 8 and by phagocytic capacity. Secretion of tumor necrosis factor alpha (TNFalpha) in response to MSU was measured by enzyme-linked immunosorbent assay. RESULTS: The panel of monocyte/macrophage cell lines revealed a close linkage between the state of differentiation and the capacity of the cells to ingest MSU crystals. TNFalpha production, however, was not linked to phagocytic ability. Peak TNFalpha levels were synthesized by cells at an intermediate state of differentiation (3.2-14.1 ng/ml), whereas mature macrophages, which efficiently phagocytosed crystals, did not secrete TNFalpha. Mature cell lines produced TNFalpha when stimulated with zymosan (5.9-6.2 ng/ml), but this was abolished by coincubation with MSU crystals. Suppression of the zymosan response was not due to apoptosis or steric hindrance by MSU crystals. Culture supernatants from mature macrophages did not stimulate endothelial cell activation, in contrast to MSU-treated cells at an earlier stage of differentiation, which stimulated intercellular adhesion molecule 1 expression on sEND endothelioma cells through the release of TNFalpha (inhibited 80.6% by anti-TNFa). CONCLUSION: We demonstrated that phagocytosis and TNFalpha production are distinct events in the response of mononuclear phagocytes to urate crystals, and these events can be distinguished at the level of macrophage differentiation. The noninflammatory removal of urate crystals by mature macrophages defines a new pathway that may be important in controlling the development of acute gout in patients with hyperuricemia.