RESUMEN
BACKGROUND: Malaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host's response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65. METHODS: C57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer. RESULTS: Plasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia. CONCLUSION: Data here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Hemo-Oxigenasa 1/farmacología , Malaria/complicaciones , Proteínas de la Membrana/farmacología , Sustancias Protectoras/farmacología , Trombocitopenia/tratamiento farmacológico , Lesión Pulmonar Aguda/etiología , Animales , Líquido del Lavado Bronquioalveolar/química , Femenino , Recuento de Leucocitos , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/fisiología , Trombocitopenia/etiologíaRESUMEN
Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDß2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDß2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement.
Asunto(s)
Antígenos CD11/metabolismo , Cadenas alfa de Integrinas/metabolismo , Malaria/fisiopatología , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Edema Encefálico/fisiopatología , Antígenos CD11/fisiología , Cloroquina/metabolismo , Modelos Animales de Enfermedad , Inflamación/metabolismo , Cadenas alfa de Integrinas/fisiología , Integrinas/inmunología , Integrinas/metabolismo , Recuento de Leucocitos , Leucocitos/metabolismo , Leucocitos/fisiología , Macrófagos/metabolismo , Malaria/genética , Malaria Cerebral/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium berghei/metabolismoRESUMEN
It is proposed that the impaired sympathoadrenal response to hypoglycemia induced by recurrent insulin-induced hypoglycemia (RH) is an adaptive phenomenon induced by specific changes in microRNA expression in the ventromedial hypothalamus (VMH). To test this hypothesis, genome-wide microRNAomic profiling of the VMH by RNA-sequencing was performed in control rats and rats treated for RH. Differential expression analysis identified microRNA-7a-5p and microRNA-665 as potential mediators of this phenomenon. To further test this hypothesis, experiments were conducted consisting of targeted lentiviral-mediated overexpression of microRNA-7a-5p and downregulation of microRNA-665 in the VMH. Hyperinsulinemic hypoglycemic clamp experiments demonstrated that targeted overexpression of microRNA-7a-5p (but not downregulation of microRNA-665) in the VMH of RH rats restored the epinephrine response to hypoglycemia. This restored response to hypoglycemia was associated with a restoration of GABAA receptor gene expression. Finally, a direct interaction of microRNA-7a-5p with the 3'-UTR of GABAA receptor α1-subunit (Gabra1) gene was demonstrated in a luciferase assay. These findings indicate that (a) the impaired sympathoadrenal response RH induces is associated with changes in VMH microRNA expression and (b) microRNA-7a-5p, possibly via direct downregulation of GABA receptor gene expression, may serve as a mediator of the altered sympathoadrenal response to hypoglycemia.
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Hipoglucemia/inducido químicamente , Hipoglucemiantes/efectos adversos , Insulina/efectos adversos , MicroARNs/metabolismo , Receptores de GABA-A/genética , Núcleo Hipotalámico Ventromedial/fisiopatología , Regiones no Traducidas 3'/genética , Adaptación Fisiológica/genética , Animales , Glucemia/análisis , Modelos Animales de Enfermedad , Regulación hacia Abajo , Epinefrina/sangre , Epinefrina/metabolismo , Retroalimentación Fisiológica , Perfilación de la Expresión Génica , Técnica de Clampeo de la Glucosa , Humanos , Hipoglucemia/sangre , Hipoglucemia/fisiopatología , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Masculino , MicroARNs/genética , Norepinefrina/sangre , Norepinefrina/metabolismo , Ratas , Ratas Sprague-Dawley , Recurrencia , Análisis de Secuencia de ARN , Sistema Nervioso Simpático/fisiopatología , Núcleo Hipotalámico Ventromedial/metabolismoRESUMEN
Insulin signaling in the central nervous system influences satiety, counterregulation, and peripheral insulin sensitivity. Neurons expressing the Glut4 glucose transporter influence peripheral insulin sensitivity. Here, we analyzed the effects of insulin receptor (IR) signaling in hypothalamic Glut4 neurons on glucose sensing as well as leptin and amino acid signaling. By measuring electrophysiological responses to low glucose conditions, we found that the majority of Glut4 neurons in the ventromedial hypothalamus (VMH) were glucose excitatory neurons. GLUT4-Cre-driven insulin receptor knockout mice with a combined ablation of IR in Glut4-expressing tissues showed increased counterregulatory response to either 2-deoxyglucose-induced neuroglycopenia or systemic insulin-induced hypoglycemia. The latter response was recapitulated in mice with decreased VMH IR expression, suggesting that the effects on the counterregulatory response are likely mediated through the deletion of IRs on Glut4 neurons in the VMH. Using immunohistochemistry in fluorescently labeled hypothalamic Glut4 neurons, we showed that IR signaling promoted hypothalamic cellular signaling responses to the rise of insulin, leptin, and amino acids associated with feeding. We concluded that hypothalamic Glut4 neurons modulated the glucagon counterregulatory response and that IR signaling in Glut4 neurons was required to integrate hormonal and nutritional cues for the regulation of glucose metabolism.
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Transportador de Glucosa de Tipo 4/fisiología , Receptor de Insulina/fisiología , Núcleo Hipotalámico Ventromedial/fisiología , Animales , Glucagón/sangre , Glucosa/metabolismo , Hipoglucemia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
It is proposed that the impaired counterregulatory response (CRR) to hypoglycemia in insulin-deficient diabetes may be due to chronic brain insulin deficiency. To test this hypothesis, streptozotocin-induced diabetic Sprague-Dawley rats were infused with insulin (3 mU/day) or artificial cerebrospinal fluid (aCSF) bilaterally into the ventromedial hypothalamus (VMH) for 2 wk and compared with nondiabetic rats. Rats underwent hyperinsulinemic (50 mU·kg-1·min-1)-hypoglycemic (~45 mg/dl) clamps. Diabetic rats demonstrated an impaired CRR to hypoglycemia, noted by a high glucose infusion rate and blunted epinephrine and glucagon responses. The defective sympathoadrenal response was restored by chronic infusion of insulin into the VMH. Diabetic rats had decreased VMH Akt phosphorylation and decreased VMH glucose transporter 4 (GLUT4) content, which was also restored by chronic infusion of insulin into the VMH. Separate experiments in nondiabetic rats in which GLUT4 translocation into the VMH was inhibited with an infusion of indinavir were notable for an impaired CRR to hypoglycemia, indicated by increased glucose infusion rate and diminished epinephrine and glucagon responses. Results suggest that, in this model of diabetes, VMH insulin deficiency impairs the sympathoadrenal response to hypoglycemia and that chronic infusion of insulin into the VMH is sufficient to normalize the sympathoadrenal response to hypoglycemia via restoration of GLUT4 expression in the VMH.
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Glucemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Hipoglucemia/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Núcleo Hipotalámico Ventromedial/efectos de los fármacos , Animales , Epinefrina/sangre , Glucagón/sangre , Técnica de Clampeo de la Glucosa , Masculino , Ratas , Ratas Sprague-Dawley , Núcleo Hipotalámico Ventromedial/metabolismoRESUMEN
ß2 integrins are critical in host defense responses to invading pathogens and inflammation. Previously, we reported that genetic deficiency of integrin αDß2 in mice altered outcomes in experimental systemic infections including accelerated mortality in animals infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of αDß2 results in impaired accumulation of leukocytes in response to peritoneal infection by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process triggered by Salmonella. Salmonella-infected animals deficient in αDß2 had increased levels of peritoneal cytokines in addition to other markers of pyroptosis, which may contribute to inflammatory injury and increased mortality in the context of impaired bacterial killing. These observations indicate important contributions of leukocyte integrins to the host response in experimental Salmonella infection and reveal previous activities of αDß2 in bacterial infection.
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Antígenos CD11/metabolismo , Antígenos CD18/metabolismo , Cadenas alfa de Integrinas/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/inmunología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno/inmunología , Recuento de Leucocitos , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , Piroptosis/inmunología , Infecciones por Salmonella/microbiologíaRESUMEN
People with insulin-treated diabetes are uniquely at risk for severe hypoglycemia-induced brain damage. Because calcium influx may mediate brain damage, we tested the hypothesis that the calcium-channel blocker, verapamil, would significantly reduce brain damage and cognitive impairment caused by severe hypoglycemia. Sprague-Dawley rats (10 weeks old) were randomly assigned to one of three treatments: 1) control hyperinsulinemic (200 mU â kg-1 â min-1)-euglycemic (80-100 mg/dL) clamps (n = 14), 2) hyperinsulinemic-hypoglycemic (10-15 mg/dL) clamps (n = 16), or 3) hyperinsulinemic-hypoglycemic clamps, followed by a single treatment with verapamil (20 mg/kg) (n = 11). Compared with euglycemic controls, hypoglycemia markedly increased dead/dying neurons in the hippocampus by 16-fold and cortex by 14-fold. Verapamil treatment strikingly decreased hypoglycemia-induced hippocampal and cortical damage, by 87% and 94%, respectively. Morris Water Maze probe trial results demonstrated that hypoglycemia induced a retention, but not encoding, memory deficit (noted by both abolished target quadrant preference and reduced target quadrant time). Verapamil treatment significantly rescued spatial memory as noted by restoration of target quadrant preference and target quadrant time. In summary, a one-time treatment with verapamil after severe hypoglycemia prevented neural damage and memory impairment caused by severe hypoglycemia. For people with insulin-treated diabetes, verapamil may be a useful drug to prevent hypoglycemia-induced brain damage.
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Encéfalo/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Hipoglucemia/complicaciones , Hipoglucemia/prevención & control , Hipoglucemiantes/uso terapéutico , Verapamilo/uso terapéutico , Animales , Glucemia/efectos de los fármacos , Encéfalo/fisiología , Insulina/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Blood vessel wall damage often results in the formation of a fibrin clot that traps inflammatory cells, including monocytes. The effect of clot formation and subsequent lysis on the expression of monocyte-derived genes involved in the development and progression of ischemic stroke and other vascular diseases, however, is unknown. Determine whether clot formation and lysis regulates the expression of human monocyte-derived genes that modulate vascular diseases. APPROACH AND RESULTS: We performed next-generation RNA sequencing on monocytes extracted from whole blood clots and using a purified plasma clot system. Numerous mRNAs were differentially expressed by monocytes embedded in clots compared with unclotted controls, and IL-8 (interleukin 8) and MCP-1 (monocyte chemoattractant protein-1) were among the upregulated transcripts in both models. Clotted plasma also increased expression of IL-8 and MCP-1, which far exceeded responses observed in lipopolysaccharide-stimulated monocytes. Upregulation of IL-8 and MCP-1 occurred in a thrombin-independent but fibrin-dependent manner. Fibrinolysis initiated shortly after plasma clot formation (ie, 1-2 hours) reduced the synthesis of IL-8 and MCP-1, whereas delayed fibrinolysis was far less effective. Consistent with these in vitro models, monocytes embedded in unresolved thrombi from patients undergoing thrombectomy stained positively for IL-8 and MCP-1. CONCLUSIONS: These findings demonstrate that clots are potent inducers of monocyte gene expression and that timely fibrinolysis attenuates inflammatory responses, specifically IL-8 and MCP-1. Dampening of inflammatory gene expression by timely clot lysis may contribute to the clinically proven efficacy of fibrinolytic drug treatment within hours of stroke onset.
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Coagulación Sanguínea/fisiología , Quimiocina CCL2/genética , Expresión Génica , Interleucina-8/genética , Monocitos/metabolismo , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/fisiopatología , Quimiocina CCL2/biosíntesis , Humanos , Interleucina-8/biosíntesis , Accidente Cerebrovascular/tratamiento farmacológico , Terapia Trombolítica , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Transcripción GenéticaRESUMEN
Integrin-targeting peptide RGDfK-labeled gold nanorods (GNR) seek to improve hyperthermia targeted to solid tumors by exploiting the known up-regulation of integrin αvß3 cell membrane proteins on solid tumor vasculature surfaces. Tumor binding specificity might be expected since surrounding tissues and endothelial cells have limited numbers of these receptors. However, RGD peptide binding to many proteins is promiscuous, with known affinity to several families of cell integrin receptors, and also possible binding to platelets after intravenous infusion via a different integrin receptor, αIIbß3, on platelets. Binding of RGDfK-targeted GNR could considerably impact platelet function, ultimately leading to increased risk of bleeding or thrombosis depending on the degree of interaction. We sought to determine if RGDfK-labeled GNR could interact with platelets and alter platelet function. Targeted and untargeted nanorods exhibited little interaction with resting platelets in platelet rich plasma (PRP) preparations. However, upon platelet activation, peptide-targeted nanorods bound actively to platelets. Addition of RGDfK-GNR to unactivated platelets had little effect on markers of platelet activation, indicating that RGDfK-nanorods were incapable of inducing platelet activation. We next tested whether activated platelet function was altered in the presence of peptide-targeted nanorods. Platelet aggregation in whole blood and PRP in the presence of targeted nanorods had no significant effect on platelet aggregation. These data suggest that RGDfK-GNR alone have little impact on platelet function in plasma. However, nonspecific nanorod binding may occur in vascular beds where activated platelets are normally cleared, such as the spleen and liver, producing a possible toxicity risk for these nanomaterials. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 209-217, 2017.
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Plaquetas/metabolismo , Oro , Nanopartículas del Metal/química , Nanotubos/química , Oligopéptidos , Activación Plaquetaria/efectos de los fármacos , Adulto , Plaquetas/ultraestructura , Femenino , Oro/química , Oro/farmacología , Humanos , Masculino , Nanopartículas del Metal/ultraestructura , Nanotubos/ultraestructura , Oligopéptidos/química , Oligopéptidos/farmacologíaRESUMEN
Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.
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Proteínas Sanguíneas/fisiología , Trampas Extracelulares/metabolismo , Neutrófilos/metabolismo , Animales , Células Cultivadas , Ensamble y Desensamble de Cromatina , Sangre Fetal/metabolismo , Histonas/metabolismo , Humanos , Recién Nacido , Inflamación/inmunología , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , Proteínas de Neoplasias/fisiología , Neutrófilos/inmunología , Procesamiento Proteico-PostraduccionalRESUMEN
BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a potentially lethal complication of clinical malaria. Acute lung injury in MA-ARDS shares features with ARDS triggered by other causes, including alveolar inflammation and increased alveolar-capillary permeability, leading to leak of protein-rich pulmonary oedema fluid. Mechanisms and physiologic alterations in MA-ARDS can be examined in murine models of this syndrome. Integrin αDß2 is a member of the leukocyte, or ß2 (CD18), sub-family of integrins, and emerging observations indicate that it has important activities in leukocyte adhesion, accumulation and signalling. The goal was to perform analysis of the lungs of mice wild type C57Bl/6 (a D (+/+) ) and Knockout C57Bl/6 (a D (-/-) ) with malaria-associated acute lung injury to better determine the relevancy of the murine models and investigate the mechanism of disease. METHODS: C57BL/6 wild type (a D (+/+) ) and deficient for CD11d sub-unit (a D (-/-) ) mice were monitored after infection with 10(5) Plasmodium berghei ANKA. CD11d subunit expression RNA was measured by real-time polymerase chain reaction, vascular barrier integrity by Evans blue dye (EBD) exclusion and cytokines by ELISA. Protein and leukocytes were measured in bronchoalveolar lavage fluid (BALF) samples. Tissue cellularity was measured by the point-counting technique, F4/80 and VCAM-1 expression by immunohistochemistry. Respiratory function was analysed by non-invasive BUXCO and mechanical ventilation. RESULTS: Alveolar inflammation, vascular and interstitial accumulation of monocytes and macrophages, and disrupted alveolar-capillary barrier function with exudation of protein-rich pulmonary oedema fluid were present in P. berghei-infected wild type mice and were improved in αDß2-deficient animals. Key pro-inflammatory cytokines were also decreased in lung tissue from α D (-/-) mice, providing a mechanistic explanation for reduced alveolar-capillary inflammation and leak. CONCLUSIONS: The results indicate that αDß2 is an important inflammatory effector molecule in P. berghei-induced MA-ARDS, and that leukocyte integrins regulate critical inflammatory and pathophysiologic events in this model of complicated malaria. Genetic deletion of integrin subunit αD in mice, leading to deficiency of integrin αDß2, alters lung inflammation and acute lung injury in a mouse model of MA-ARDS caused by P. berghei.
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Antígenos CD11/metabolismo , Cadenas alfa de Integrinas/metabolismo , Malaria/complicaciones , Síndrome de Dificultad Respiratoria/patología , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Citocinas/análisis , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Azul de Evans/metabolismo , Perfilación de la Expresión Génica , Inmunohistoquímica , Recuento de Leucocitos , Pulmón/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Permeabilidad , Plasmodium berghei/crecimiento & desarrollo , Proteínas/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Pruebas de Función RespiratoriaRESUMEN
Integrin α(D)ß(2) is the most recently identified member of the leukocyte, or ß(2), subfamily of integrin heterodimers. Its distribution and functions on human leukocytes have not been clearly defined and are controversial. We examined these issues and found that α(D)ß(2) is prominently expressed by leukocytes in whole blood from healthy human subjects, including most polymorphonuclear leukocytes and monocytes. We also found that α(D)ß(2) is displayed by leukocytes in the alveoli of uninjured and inflamed human lungs and by human monocyte-derived macrophages and dendritic cells, indicating broad myeloid expression. Using freshly-isolated human monocytes, we found that α(D)ß(2) delivers outside-in signals to pathways that regulate cell spreading and gene expression. Screening expression analysis followed by validation of candidate transcripts demonstrated that engagement of α(D)ß(2) induces mRNAs encoding inflammatory chemokines and cytokines and secretion of their protein products. Thus, α(D)ß(2) is a major member of the integrin repertoire of both circulating and tissue myeloid leukocytes in humans. Its broad expression and capacity for outside-in signaling indicate that it is likely to have important functions in clinical syndromes of infection, inflammation, and tissue injury.
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Antígenos CD11/metabolismo , Inflamación/metabolismo , Cadenas alfa de Integrinas/metabolismo , Leucocitos/metabolismo , Transducción de Señal , Diferenciación Celular , Citometría de Flujo , HumanosRESUMEN
Dengue is the most prevalent human arbovirus disease in the world. Dengue infection has a large spectrum of clinical manifestations, from self-limited febrile illness to severe syndromes accompanied by bleeding and shock. Thrombocytopenia and vascular leak with altered cytokine profiles in plasma are features of severe dengue. Although monocytes have been recognized as important sources of cytokines in dengue, the contributions of platelet-monocyte interactions to inflammatory responses in dengue have not been addressed. Patients with dengue were investigated for platelet-monocyte aggregate formation. Platelet-induced cytokine responses by monocytes and underlying mechanisms were also investigated in vitro. We observed increased levels of platelet-monocyte aggregates in blood samples from patients with dengue, especially patients with thrombocytopenia and increased vascular permeability. Moreover, the exposure of monocytes from healthy volunteers to platelets from patients with dengue induced the secretion of the cytokines IL-1ß, IL-8, IL-10 and MCP-1, whereas exposure to platelets from healthy volunteers only induced the secretion of MCP-1. In addition to the well-established modulation of monocyte cytokine responses by activated platelets through P-selectin binding, we found that interaction of monocytes with apoptotic platelets mediate IL-10 secretion through phosphatidylserine recognition in platelet-monocyte aggregates. Moreover, IL-10 secretion required platelet-monocyte contact but not phagocytosis. Together, our results demonstrate that activated and apoptotic platelets aggregate with monocytes during dengue infection and signal specific cytokine responses that may contribute to the pathogenesis of dengue.
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Plaquetas/inmunología , Dengue/inmunología , Monocitos/inmunología , Activación Plaquetaria/inmunología , Adulto , Apoptosis/inmunología , Permeabilidad Capilar , Quimiocina CCL2/metabolismo , Virus del Dengue/inmunología , Femenino , Humanos , Inflamación/inmunología , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Masculino , Selectina-P/inmunología , Fagocitosis , Fosfatidilserinas/inmunología , Trombocitopenia/inmunologíaRESUMEN
Allergic asthma is a chronic inflammatory airway disease with increasing prevalence around the world. Current asthma therapy includes drugs that usually cause significant side effects, justifying the search for new anti-asthmatic drugs. Curine is a bisbenzylisoquinoline alkaloid that modulates calcium influx in many cell types; however, its anti-allergic and putative toxic effects remain to be elucidated. Our aim was to investigate the effects of curine on eosinophil activation and airway hyper-responsiveness (AHR) and to characterize its potential toxic effects. We used a mouse model of allergic asthma induced by sensitization and challenge with ovalbumin (OVA) to evaluate the anti-allergic effects of oral treatment with curine. The oral administration of curine significantly inhibited eosinophilic inflammation, eosinophil lipid body formation and AHR in animals challenged with OVA compared with animals in the untreated group. The curine treatment also reduced eotaxin and IL-13 production triggered by OVA. Verapamil, a calcium channel antagonist, had similar anti-allergic properties, and curine pre-treatment inhibited the calcium-induced tracheal contractile response ex-vivo, suggesting that the mechanism by which curine exerts its effects is through the inhibition of a calcium-dependent response. A toxicological evaluation showed that orally administered curine did not significantly alter the biochemical, hematological, behavioral and physical parameters measured in the experimental animals compared with saline-treated animals. In conclusion, curine showed anti-allergic activity through mechanisms that involve inhibition of IL-13 and eotaxin and of Ca(++) influx, without inducing evident toxicity and as such, has the potential for the development of anti-asthmatic drugs.
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Antiasmáticos/toxicidad , Asma/tratamiento farmacológico , Eosinófilos/efectos de los fármacos , Isoquinolinas/toxicidad , Administración Oral , Animales , Hiperreactividad Bronquial/tratamiento farmacológico , Calcio/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/metabolismo , Inflamación/tratamiento farmacológico , Interleucina-13/antagonistas & inhibidores , Interleucina-13/metabolismo , Masculino , Menispermaceae/química , Ratones , Ratones Endogámicos BALB C , Nivel sin Efectos Adversos Observados , Ovalbúmina/metabolismo , Ratas , Ratas Wistar , Verapamilo/farmacologíaRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) induces a pro-thrombotic and pro-inflammatory milieu. Although timely antibiotic administration in MRSAsepsis may improve outcomes by arresting bacterial growth, the effects of antibiotics on mitigating injurious thrombo-inflammatory cellular responses remains unexplored. Using a newly developed human whole blood model and an in vivo mouse model of MRSAinfection, we examined how antibiotics inhibit MRSAinduced thrombo-inflammatory pathways. Human whole blood was inoculated with MRSA. Thrombin generation and inflammatory cytokine synthesis was measured in the presence or absence of linezolid and vancomycin. C57BL/6 mice were injected with MRSA and the effect of vancomycin administration was examined. MRSAaccelerated thrombin generation in a time- and concentration-dependent manner andinduced the release of cytokines, including interleukin (IL)-6, IL-8, and monocyte chemotactic protein (MCP)-1. The increase in thrombin generation and inflammatory responses was mediated through the synthesis of tissue factor and cytokines, respectively, and the release of microparticles. The early administration of antibiotics restored normal thrombin generation patterns and significantly reduced the synthesis of cytokines. In contrast, when antibiotic administration was delayed, thrombin generation and cytokine synthesis were not significantly reduced. In mice infected with MRSA, early antibiotic administration reduced thrombin anti-thrombin complexes and cytokine synthesis, whereas delayed antibiotic administration did not. These data provide novel mechanistic evidence of the importance of prompt antibiotic administration in infectious syndromes.
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Acetamidas/administración & dosificación , Antibacterianos/administración & dosificación , Inflamación/prevención & control , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Oxazolidinonas/administración & dosificación , Sepsis/tratamiento farmacológico , Infecciones Estafilocócicas/tratamiento farmacológico , Trombosis/prevención & control , Vancomicina/administración & dosificación , Animales , Coagulación Sanguínea/efectos de los fármacos , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/inmunología , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/microbiología , Citocinas/sangre , Esquema de Medicación , Humanos , Inflamación/sangre , Inflamación/inmunología , Inflamación/microbiología , Mediadores de Inflamación/sangre , Linezolid , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Staphylococcus aureus Resistente a Meticilina/inmunología , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/microbiología , Sepsis/sangre , Sepsis/inmunología , Sepsis/microbiología , Infecciones Estafilocócicas/sangre , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Trombina/metabolismo , Trombosis/sangre , Trombosis/inmunología , Trombosis/microbiología , Factores de TiempoRESUMEN
Bacteria can enter the bloodstream in response to infectious insults. Bacteremia elicits several immune and clinical complications, including thrombocytopenia. A primary cause of thrombocytopenia is shortened survival of platelets. We demonstrate that pathogenic bacteria induce apoptotic events in platelets that include calpain-mediated degradation of Bcl-x(L), an essential regulator of platelet survival. Specifically, bloodstream bacterial isolates from patients with sepsis induce lateral condensation of actin, impair mitochondrial membrane potential, and degrade Bcl-x(L) protein in platelets. Bcl-x(L) protein degradation is enhanced when platelets are exposed to pathogenic Escherichia coli that produce the pore-forming toxin α-hemolysin, a response that is markedly attenuated when the gene is deleted from E coli. We also found that nonpathogenic E coli gain degrading activity when they are forced to express α-hemolysin. Like α-hemolysin, purified α-toxin readily degrades Bcl-x(L) protein in platelets, as do clinical Staphylococcus aureus isolates that produce α-toxin. Inhibition of calpain activity, but not the proteasome, rescues Bcl-x(L) protein degradation in platelets coincubated with pathogenic E coli including α-hemolysin producing strains. This is the first evidence that pathogenic bacteria can trigger activation of the platelet intrinsic apoptosis program and our results suggest a new mechanism by which bacterial pathogens might cause thrombocytopenia in patients with bloodstream infections.
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Plaquetas/microbiología , Escherichia coli/fisiología , Interacciones Huésped-Patógeno , Staphylococcus aureus/fisiología , Proteína bcl-X/metabolismo , Apoptosis , Plaquetas/citología , Plaquetas/metabolismo , Calpaína/metabolismo , Infecciones por Escherichia coli/microbiología , Humanos , Proteolisis , Infecciones Estafilocócicas/microbiologíaRESUMEN
BACKGROUND: Cissampelos sympodialis Eichl. (Menispermaceae) is a plant found in Northeastern and Southeast of Brazil and hot water infusion of C. sympodialis root bark is largely used in the indigenous and folk medicine to treat several inflammatory disorders, including asthma. Asthma is a chronic inflammatory allergic disease characterized by airway hyperreactivity (AHR), eosinophil tissue infiltration and lung remodeling. The aim of this study was to evaluate the therapeutic effect of C. sympodialis and its isolated alkaloid warifteine on allergen triggered airway hyperreactivity (AHR) and lung remodeling in murine model of asthma. METHODOLOGY/PRINCIPAL FINDINGS: The oral pre-treatment with C. sympodialis or warifteine inhibited allergen-induced AHR to inhaled methacholine and IL-13 levels in the bronchoalveolar lavage (BAL). In order to investigate the therapeutic potential of C. sympodialis and warifteine, animals were treated 1h after the last ovalbumin (OVA) challenge in sensitized animals. Similarly to the pre-treatment, post-treatment with warifteine was effective to inhibit significantly AHR to inhaled methacholine and to reduce IL-13 levels in the BAL. In addition, oral pre- or post-treatments with C. sympodialis or warifteine reduced OVA-induced eosinophil tissue infiltration, mucus production and subepithelial fibrosis to values similar to nonallergic controls. CONCLUSIONS: Our data show the anti-allergic and immunoregulatory properties of C. sympodialis, acting mostly through the active compound warifteine, to inhibit the airway hyperreactivity and lung remodeling through a mechanism at least partially dependent of IL-13 and eosinophil inhibition. Therefore placing warifteine as an interesting therapeutic candidate in allergic inflammation and corroborating the folk medicine use of C. sympodialis as anti-allergic plant.
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Alcaloides/uso terapéutico , Asma/tratamiento farmacológico , Cissampelos/química , Fitoterapia , Alcaloides/química , Animales , Asma/inmunología , Asma/patología , Asma/fisiopatología , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/fisiopatología , Líquido del Lavado Bronquioalveolar/inmunología , Colágeno/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/efectos de los fármacos , Eosinófilos/patología , Femenino , Humanos , Interleucina-13/biosíntesis , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Plantas Medicinales/química , Hipersensibilidad Respiratoria/tratamiento farmacológico , Hipersensibilidad Respiratoria/fisiopatologíaRESUMEN
Poly(amidoamine) (PAMAM) dendrimers have been proposed for a variety of biomedical applications and are increasingly studied as model nanomaterials for such use. The dendritic structure features both modular synthetic control of molecular size and shape and presentation of multiple equivalent terminal groups. These properties make PAMAM dendrimers highly functionalizable, versatile single-molecule nanoparticles with a high degree of consistency and low polydispersity. Recent nanotoxicological studies showed that intravenous administration of amine-terminated PAMAM dendrimers to mice was lethal, causing a disseminated intravascular coagulation-like condition. To elucidate the mechanisms underlying this coagulopathy, in vitro assessments of platelet functions in contact with PAMAM dendrimers were undertaken. This study demonstrates that cationic G7 PAMAM dendrimers activate platelets and dramatically alter their morphology. These changes to platelet morphology and activation state substantially altered platelet function, including increased aggregation and adherence to surfaces. Surprisingly, dendrimer exposure also attenuated platelet-dependent thrombin generation, indicating that not all platelet functions remained intact. These findings provide additional insight into PAMAM dendrimer effects on blood components and underscore the necessity for further research on the effects and mechanisms of PAMAM-specific and general nanoparticle toxicity in blood.
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Plaquetas/efectos de los fármacos , Dendrímeros/efectos adversos , Plaquetas/metabolismo , Células Cultivadas , Citometría de Flujo , Humanos , Microscopía Confocal , Nanopartículas/efectos adversos , Nanotecnología , Activación Plaquetaria/efectos de los fármacos , Pruebas de Función Plaquetaria , Trombina/metabolismoRESUMEN
Platelets are chief effector cells in hemostasis. In addition, however, their specializations include activities and intercellular interactions that make them key effectors in inflammation and in the continuum of innate and adaptive immunity. This review focuses on the immune features of human platelets and platelets from experimental animals and on interactions between inflammatory, immune, and hemostatic activities of these anucleate but complex and versatile cells. The experimental findings and evidence for physiologic immune functions include previously unrecognized biologic characteristics of platelets and are paralleled by new evidence for unique roles of platelets in inflammatory, immune, and thrombotic diseases.
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Inmunidad Adaptativa , Plaquetas/inmunología , Hemostasis/inmunología , Inmunidad Innata , Inflamación/inmunología , Animales , Humanos , Trombosis/inmunologíaRESUMEN
Members of the nuclear factor of activated T cell (NFAT) family of transcription factors were originally described in T lymphocytes but later shown to be expressed in several immune and non-immune cell types. NFAT proteins can modulate cellular transformation intrinsically, and NFAT-deficient (NFAT1-/-) mice are indeed more susceptible to transformation than wild-type counterparts. However, the contribution of an NFAT1-/- microenvironment to tumor progression has not been studied. We have addressed this question by inoculating NFAT1-/- mice with B16F10 melanoma cells intravenously, an established model of tumor homing and growth. Surprisingly, NFAT1-/- animals sustained less tumor growth in the lungs after melanoma inoculation than wild-type counterparts. Even though melanoma cells equally colonize NFAT1-/- and wild-type lungs, tumors do not progress in the absence of NFAT1 expression. A massive mononuclear perivascular infiltrate and reduced expression of TGF-ß in the absence of NFAT1 suggested a role for tumor-infiltrating immune cells and the cytokine milieu. However, these processes are independent of an IL-4-induced regulatory tumor microenvironment, since lack of this cytokine does not alter the phenotype in NFAT1-/- animals. Bone marrow chimera experiments meant to differentiate the contributions of stromal and infiltrating cells to tumor progression demonstrated that NFAT1-induced susceptibility to pulmonary tumor growth depends on NFAT1-expressing parenchyma rather than on bone marrow-derived cells. These results suggest an important role for NFAT1 in radio-resistant tumor-associated parenchyma, which is independent of the anti-tumor immune response and Th1 versus Th2 cytokine milieu established by the cancer cells, but able to promote site-specific tumor growth.