RESUMEN
Intestinal fibrosis is a common complication that affects more than 50% of Crohn´s Disease (CD) patients. There is no pharmacological treatment against this complication, with surgery being the only option. Due to the unknown role of P2X7 in intestinal fibrosis, we aim to analyze the relevance of this receptor in CD complications. Surgical resections from CD and non-Inflammatory Bowel Disease (IBD) patients were obtained. Intestinal fibrosis was induced with two different murine models: heterotopic transplant model and chronic-DSS colitis in wild-type and P2X7-/- mice. Human small intestine fibroblasts (HSIFs) were transfected with an siRNA against P2X7 and treated with TGF-ß. A gene and protein expression of P2X7 receptor was significantly increased in CD compared to non-IBD patients. The lack of P2X7 in mice provoked an enhanced collagen deposition and increased expression of several profibrotic markers in both murine models of intestinal fibrosis. Furthermore, P2X7-/- mice exhibited a higher expression of proinflammatory cytokines and a lower expression of M2 macrophage markers. Moreover, the transient silencing of the P2X7 receptor in HSIFs significantly induced the expression of Col1a1 and potentiated the expression of Col4 and Col5a1 after TGF-ß treatment. P2X7 regulates collagen expression in human intestinal fibroblasts, while the lack of this receptor aggravates intestinal fibrosis.
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Fibroblastos , Intestinos , Receptores Purinérgicos P2X7 , Animales , Humanos , Ratones , Colitis/metabolismo , Colitis/patología , Colágeno/genética , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Fibroblastos/metabolismo , Intestinos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND: Phosphatidylserine (PS) externalization out of the membrane facilitates the eryptotic erythrocytes (EE) binding to endothelial cells (EC), potentially leading to atherosclerosis. Thus, the levels of eryptosis and interactions of EE-EC in hypercholesterolemic patients, either non-medicated or medicated, compared with healthy subjects were studied. METHODS: A total of 56 subjects clustered into three groups: (control (n = 20), hypercholesterolemic non-treated (HCNT) (n = 15), and statin-treated (HCT) (n = 21)) were enrolled in this cross-sectional study. Biochemical parameters were determined with validated and standard methods. PS exposure was estimated from annexin-V-binding, cell volume from forward scatter (FSC), and GSH from CMFDA fluorescence by flow cytometry. The erythrocyte-EC adhesion assay was performed by using the parallel-plate flow chamber technique. RESULTS: Higher PS externalization and adhesion of erythrocytes to EC (P < .05) was found in hypercholesterolemic subjects, regardless of statin treatment, compared with the control group. Although no correlation between FSC and PS externalization with other parameters was found, GSH was inversely correlated with erythrocyte adhesion, which was significantly correlated with total cholesterol, LDL-c, and apolipoprotein B. CONCLUSION: The link between hypercholesterolemia and eryptosis suggests a possible detrimental impact of this binomial on endothelial function with possible further development of atherosclerosis and microcirculation problems in hypercholesterolemic patients, independently of statin therapy.
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Eriptosis , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Calcio , Estudios Transversales , Células Endoteliales , Endotelio , Eritrocitos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéuticoRESUMEN
Cardiovascular diseases are a leading cause of death. Blood-cell interactions and endothelial dysfunction are fundamental in thrombus formation, and so further knowledge of the pathways involved in such cellular crosstalk could lead to new therapeutical approaches. Neutrophils are secretory cells that release well-known soluble inflammatory signaling mediators and other complex cellular structures whose role is not fully understood. Studies have reported that neutrophil extracellular vesicles (EVs) and neutrophil extracellular traps (NETs) contribute to thrombosis. The objective of this review is to study the role of EVs and NETs as key factors in the transition from inflammation to thrombosis. The neutrophil secretome can promote thrombosis due to the presence of different factors in the EVs bilayer that can trigger blood clotting, and to the release of soluble mediators that induce platelet activation or aggregation. On the other hand, one of the main pathways by which NETs induce thrombosis is through the creation of a scaffold to which platelets and other blood cells adhere. In this context, platelet activation has been associated with the induction of NETs release. Hence, the structure and composition of EVs and NETs, as well as the feedback mechanism between the two processes that causes pathological thrombus formation, require exhaustive analysis to clarify their role in thrombosis.
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Trampas Extracelulares/metabolismo , Neutrófilos/metabolismo , Trombosis/metabolismo , Animales , Plaquetas/metabolismo , Exosomas/metabolismo , Humanos , Transducción de SeñalRESUMEN
OBJECTIVE: To study leukocyte-endothelium interaction, a measure of the initial phase of atheromatosis, in children with overweight or obesity. STUDY DESIGN: A prospective study was conducted in 77 children aged 7-16 years; 47 were children with overweight/obesity and 30 were normal weight. Polymorphonuclear neutrophils (PMNs) and peripheral blood mononuclear cells were isolated from venous blood samples and the interaction of leukocytes over a monolayer of human umbilical vein endothelial cells was analyzed using flow chamber microscopy. The variables studied included leukocyte rolling velocity, rolling flux, and adhesion to endothelial cells. These were compared between children with overweight/obesity and control children. Correlation between the measures of leukocyte-endothelium interaction and anthropometric and biochemical variables was evaluated. RESULTS: In comparison with normal weight children, the PMNs and peripheral blood mononuclear cells of the overweight/obesity group showed a reduction in rolling velocity (P = .000 and P = .001, respectively) and an increase in rolling flux (P = .001 and P = .004), and adhesion (P = .003 and P = .002). The homeostasis model of insulin resistance was correlated inversely with rolling velocity and positively with rolling flux in PMNs. C-reactive protein was correlated positively with rolling flux and adhesion in both types of leucocytes. Fat mass index was correlated with all measures of leukocyte-endothelial interaction and proved to be the main predictor of leukocyte adhesion in the multiple regression analysis (P = .001 for PMNs and P = .006 for peripheral blood mononuclear cells). CONCLUSIONS: Excess fat mass in children is related to the activation of the leukocyte-endothelium interaction, potentially contributing to the development of atherosclerosis.
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Células Endoteliales/fisiología , Leucocitos Mononucleares/fisiología , Obesidad Infantil/fisiopatología , Adolescente , Proteína C-Reactiva/análisis , Estudios de Casos y Controles , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Niño , Femenino , Humanos , Resistencia a la Insulina/fisiología , Masculino , Neutrófilos/fisiología , Estudios ProspectivosRESUMEN
The P2X7 receptor (P2X7R) possesses a unique structure associated to an as yet not fully understood mechanism of action that facilitates cell permeability to large ionic molecules through the receptor itself and/or nearby membrane proteins. High extracellular adenosine triphosphate (ATP) levels-inexistent in physiological conditions-are required for the receptor to be triggered and contribute to its role in cell damage signaling. The inconsistent data on its activation pathways and the few studies performed in natively expressed human P2X7R have led us to review the structure, activation pathways, and specific cellular location of P2X7R in order to analyze its biological relevance. The ATP-gated P2X7R is a homo-trimeric receptor channel that is occasionally hetero-trimeric and highly polymorphic, with at least nine human splice variants. It is localized predominantly in the cellular membrane and has a characteristic plasticity due to an extended C-termini, which confers it the capacity of interacting with membrane structural compounds and/or intracellular signaling messengers to mediate flexible transduction pathways. Diverse drugs and a few endogenous molecules have been highlighted as extracellular allosteric modulators of P2X7R. Therefore, studies in human cells that constitutively express P2X7R need to investigate the precise endogenous mediator located nearby the activation/modulation domains of the receptor. Such research could help us understand the possible physiological ATP-mediated P2X7R homeostasis signaling.
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Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Polimorfismo Genético , Estructura Cuaternaria de Proteína , Receptores Purinérgicos P2X7/genética , Transducción de Señal , Transcripción GenéticaRESUMEN
BACKGROUND: The aim of this study was to evaluate markers of inflammation, oxidative stress and endothelial function in a disease-related malnutrition (DRM) outpatient population. METHODS: For this cross-sectional study, a total of 83 subjects were included and clustered in 3 groups: 34 with normonutrition (NN), 21 with DRM without inflammation (DRM-I) and 28 with DRM and inflammation (DRM + I). Nutritional diagnosis was conducted for all subjects according to ASPEN. Biochemical parameters, proinflammatory cytokines, reactive oxygen species production, glutathione, mitochondrial membrane potential, oxygen consumption, adhesion molecules and leukocyte-endothelium interactions were evaluated. RESULTS: DRM + I patients showed lower albumin, prealbumin, transferrin, and retinol-binding protein levels with respect to the NN group (p < 0.05), differences that were less noticeable in the DRM-I group. DRM + I was associated with a significant increase in hsCRP and IL6 vs the NN and DRM-I groups, and TNFα was increased in both DRM vs NN. DRM was characterised by increased oxidative stress, which was marked by a significant increase in ROS levels and a decrease in mitochondrial membrane potential in the DRM + I group. An evident reduction in mitochondrial oxygen consumption and glutathione concentration was observed in both DRM groups, and was accompanied by increased leukocyte adhesion and adhesion molecules and decreased rolling velocity in the DRM + I group. Furthermore, percentage of weight loss was negatively correlated with albumin, prealbumin, transferrin, O2 consumption, glutathione and leukocyte rolling velocity, and positively correlated with hsCRP, IL6, TNFα, ROS, leukocyte adhesion, and VCAM-1. CONCLUSIONS: Our results show that DRM is associated with oxidative stress and an inflammatory state, with a deterioration of endothelial dysfunction in the DRM + I population.
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Leucocitos/fisiología , Desnutrición/sangre , Desnutrición/complicaciones , Mitocondrias/fisiología , Estrés Oxidativo , Anciano , Adhesión Celular , Estudios Transversales , Citocinas/sangre , Femenino , Glutatión/sangre , Humanos , Inflamación/sangre , Inflamación/fisiopatología , Masculino , Potencial de la Membrana Mitocondrial , Persona de Mediana Edad , Oxígeno/sangre , Especies Reactivas de Oxígeno/sangre , EspañaRESUMEN
Background: The purinergic system is known to underlie prothrombotic and proinflammatory vascular programs, making the profile of experimental actions demonstrated by abacavir compatible with thrombogenesis. However, direct evidence of a prothrombotic effect by the drug has been lacking. Methods: The present study appraised the effects of abacavir in a well-validated animal model of arterial thrombosis. The role of ATP-P2X7 receptors in the actions of the drug was also assessed, and the actions of recognized vascular-damaging agents and other nucleoside reverse-transcriptase inhibitors (NRTIs) were evaluated and compared to those of abacavir. Results: Abacavir dose-dependently promoted thrombus formation. This effect was reversed by a P2X7-receptor antagonist and was nonexistent in P2X7 knockout mice. The effects of abacavir were similar to those of diclofenac and rofecoxib. Other NRTIs had no thrombosis-related effects. Conclusion: Abacavir promotes arterial thrombosis through interference with purinergic signaling, suggesting a possible biological mechanism for the clinical association of abacavir with cardiovascular diseases.
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Fármacos Anti-VIH/efectos adversos , Didesoxinucleósidos/efectos adversos , Trombosis/inducido químicamente , Animales , Fármacos Anti-VIH/administración & dosificación , Didesoxinucleósidos/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Ratones Noqueados , Receptores Purinérgicos P2X7/metabolismoRESUMEN
BACKGROUND: To evaluate the relationship between leukocyte-endothelial cell interactions and oxidative stress parameters in non-diabetic patients with different grades of obesity. MATERIAL AND METHODS: For this cross-sectional study, 225 subjects were recruited from January 1, 2014 to December 31, 2016 and divided into groups according to BMI (<30 kg/m2 , 30-40 kg/m2 and >40 kg/m²). We determined clinical parameters, systemic inflammatory markers, soluble cellular adhesion molecules, leukocyte-endothelium cell interactions-rolling flux, velocity and adhesion-, oxidative stress parameters-total ROS, total superoxide, glutathione-and mitochondrial membrane potential in leukocytes. RESULTS: We verified that HOMA-IR and hsCRP increased progressively as obesity developed, whereas A1c, IL6 and TNFα were augmented in the BMI > 40 kg/m² group. The cellular adhesion molecule sP-selectin was increased in patients with obesity, while sICAM, total ROS, total superoxide and mitochondrial membrane potential were selectively higher in the BMI > 40 kg/m² group. Obesity induced a progressive decrease in rolling velocity and an enhancement of rolling flux and leukocyte adhesion. CONCLUSION: Our findings reveal that endothelial dysfunction markers are altered in human obesity and are associated with proinflammatory cytokines and increased oxidative stress parameters.
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Células Endoteliales/fisiología , Leucocitos/fisiología , Obesidad/fisiopatología , Estrés Oxidativo/fisiología , Adolescente , Adulto , Anciano , Índice de Masa Corporal , Adhesión Celular/fisiología , Comunicación Celular/fisiología , Estudios Transversales , Citocinas/metabolismo , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/fisiología , Persona de Mediana Edad , Especies Reactivas de Oxígeno/metabolismo , Adulto JovenRESUMEN
The NNRTI efavirenz has long been one of the most frequently employed antiretroviral drugs in the multidrug regimens used to treat HIV infection, in accordance with its well-demonstrated antiretroviral efficacy and favourable pharmacokinetics. However, growing concern about its adverse effects has sometimes led to efavirenz being replaced by other drugs in the initial treatment selection or to switching of therapy to efavirenz-free regimens in experienced patients. Neurological and neuropsychiatric reactions are the manifestations most frequently experienced by efavirenz-treated patients and range from transitory effects, such as nightmares, dizziness, insomnia, nervousness and lack of concentration, to more severe symptoms including depression, suicidal ideation or even psychosis. In addition, efavirenz has recently been associated with mild/moderate neurocognitive impairment, which is of specific relevance given that half of the patients receiving ART eventually suffer some form of HIV-associated neurocognitive disorder. The mechanisms responsible for efavirenz-induced neurotoxicity are unclear, although growing evidence points to disturbances in brain mitochondrial function and bioenergetics. This review offers a comprehensive overview of the current evidence on the interaction that efavirenz displays with the CNS, including the penetration and concentration of the drug in the brain. We discuss the prevalence, types and specificities of its side effects and recently uncovered cellular mechanisms that may be involved in their development.
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Fármacos Anti-VIH/efectos adversos , Benzoxazinas/efectos adversos , Sistema Nervioso Central/efectos de los fármacos , Inhibidores Enzimáticos del Citocromo P-450/efectos adversos , Inhibidores de la Transcriptasa Inversa/efectos adversos , Alquinos , Animales , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa/efectos adversos , Benzoxazinas/farmacología , Benzoxazinas/uso terapéutico , Enfermedades del Sistema Nervioso Central/etiología , Ciclopropanos , Citocromo P-450 CYP2B6/genética , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Inhibidores Enzimáticos del Citocromo P-450/uso terapéutico , Modelos Animales de Enfermedad , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Humanos , Farmacogenética , Polimorfismo de Nucleótido Simple , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéuticoRESUMEN
BACKGROUND: Anorexia nervosa is a common psychiatric disorder in adolescence and is related to cardiovascular complications. Our aim was to study the effect of anorexia nervosa on metabolic parameters, leucocyte-endothelium interactions, adhesion molecules and proinflammatory cytokines. MATERIALS AND METHODS: This multicentre, cross-sectional, case-control study employed a population of 24 anorexic female patients and 36 controls. We evaluated anthropometric and metabolic parameters, interactions between leucocytes polymorphonuclear neutrophils (PMN) and human umbilical vein endothelial cells (HUVEC), proinflammatory cytokines such as tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and soluble cellular adhesion molecules (CAMs) including E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). RESULTS: Anorexia nervosa was related to a decrease in weight, body mass index, waist circumference, systolic blood pressure, glucose, insulin and HOMA-IR, and an increase in HDL cholesterol. These effects disappeared after adjusting for BMI. Anorexia nervosa induced a decrease in PMN rolling velocity and an increase in PMN rolling flux and PMN adhesion. Increases in IL-6 and TNF-α and adhesion molecule VCAM-1 were also observed. CONCLUSIONS: This study supports the hypothesis of an association between anorexia nervosa, inflammation and the induction of leucocyte-endothelium interactions. These findings may explain, in part at least, the increased risk of vascular disease among patients with anorexia nervosa.
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Anorexia Nerviosa/patología , Células Endoteliales/fisiología , Leucocitos Mononucleares/fisiología , Adolescente , Adulto , Índice de Masa Corporal , Estudios de Casos y Controles , Moléculas de Adhesión Celular/metabolismo , Estudios Transversales , Citocinas/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación/metabolismo , Inflamación/patología , Adulto JovenRESUMEN
BACKGROUND: Neurological pathogenesis is associated with mitochondrial dysfunction and differences in neuronal/glial handling of oxygen and glucose. The main side effects attributed to efavirenz involve the CNS, but the underlying mechanisms are unclear. METHODS: Human cell lines and rat primary cultures of neurons and astrocytes were treated with clinically relevant efavirenz concentration. RESULTS: Efavirenz alters mitochondrial respiration, enhances reactive oxygen species generation, undermines mitochondrial membrane potential, and reduces adenosine triphosphate (ATP) levels in a concentration-dependent fashion in both neurons and glial cells. However, it activates adenosine monophosphate-activated protein kinase only in glial cells, upregulating glycolysis and increasing intracellular ATP levels, which do not occur in neurons. To reproduce the conditions that often exist in human immunodeficiency virus-related neuroinflammatory disorders, the effects of efavirenz were evaluated in the presence of exogenous nitric oxide, an inflammatory mediator and mitochondrial inhibitor. The combination potentiated the effects on mitochondrial parameters in both neurons and glial cells, but ATP generation and lactate production were enhanced only in glial cells. CONCLUSIONS: Efavirenz affects the bioenergetics of neurons through a mechanism involving acute mitochondrial inhibition, an action exacerbated in neuroinflammatory conditions. A similar scenario of glial cells survival and degeneration of neurons with signs of mitochondrial dysfunction and oxidative stress has been associated with neurocognitive disorders.
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Benzoxazinas/efectos adversos , Metabolismo Energético/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/efectos adversos , Alquinos , Animales , Astrocitos/efectos de los fármacos , Benzoxazinas/farmacología , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclopropanos , Relación Dosis-Respuesta a Droga , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neuroglía/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Wistar , Inhibidores de la Transcriptasa Inversa/farmacología , Superóxidos/metabolismoRESUMEN
OBJECTIVES: The potential cardiovascular (CV) toxicity associated with combined antiretroviral therapy (cART) has been attributed mainly to the nucleoside reverse transcriptase inhibitors abacavir and didanosine. However, the other two components of cART--non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs)--may also be implicated, either directly or by influencing the action of the other drugs. This study evaluates the acute direct effects of the NNRTIs efavirenz and nevirapine and one of the most widely employed PIs, lopinavir, on leucocyte-endothelium interactions, a hallmark of CV disease. METHODS: Drugs were analysed in vitro in human cells (interactions of peripheral blood polymorphonuclear or mononuclear cells with human umbilical vein endothelial cells) using a flow chamber system, and in vivo in rat mesenteric vessels by means of intravital microscopy. The expression of adhesion molecules in leucocytes and endothelial cells was studied by flow cytometry, and the role of these molecules in white cell recruitment was evaluated by pre-treating human cells or rats with blocking antibodies. RESULTS: Efavirenz and nevirapine, but not lopinavir, increased the rolling flux and adhesion of leucocytes in vitro and in vivo while inducing emigration in rat venules. Efavirenz, but not nevirapine, augmented the levels of CD11b, CD11c and CD18 in neutrophils and monocytes. The actions of efavirenz, but not of nevirapine, were reversed by antibodies against Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18) or ICAM-1 (CD54). CONCLUSIONS: NNRTIs, but not PIs, interfere with leucocyte-endothelial interactions. However, differences between efavirenz and nevirapine suggest a specific CV profile for each compound.
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Fármacos Anti-VIH/metabolismo , Benzoxazinas/metabolismo , Adhesión Celular , Endotelio/fisiología , Integrina alfaXbeta2/metabolismo , Leucocitos/fisiología , Antígeno de Macrófago-1/metabolismo , Alquinos , Animales , Células Cultivadas , Ciclopropanos , Endotelio/efectos de los fármacos , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Leucocitos/efectos de los fármacos , Lopinavir/metabolismo , Masculino , Nevirapina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: There is controversy regarding cardiovascular (CV) toxicity of the nucleoside reverse-transcriptase inhibitors used to treat human immunodeficiency virus infection. METHODS: We evaluated the effects of nucleoside reverse-transcriptase inhibitors on leukocyte-endothelium interactions, a hallmark of CV diseases, in rat mesenteric vessels using intravital microscopy and in human arterial cells using a flow chamber system. RESULTS: Abacavir and didanosine increased rolling, adhesion and emigration in rat vessels. These effects were reversed with antibodies against Macrophage-1 antigen (Mac-1) or intercellular adhesion molecule 1 and were reproduced in human cells. Lamivudine, zidovudine, emtricitabine, and tenofovir had no effects. CONCLUSIONS: Our results support the association of abacavir and didanosine with CV diseases.
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Fármacos Anti-VIH/farmacología , Didanosina/farmacología , Didesoxinucleósidos/farmacología , Células Endoteliales/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Animales , Arteriolas/efectos de los fármacos , Arteriolas/patología , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Adhesión Celular , Comunicación Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Rodamiento de Leucocito/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Neutrófilos/metabolismo , Ratas , Ratas Sprague-Dawley , Vénulas/efectos de los fármacos , Vénulas/patologíaRESUMEN
BACKGROUND AND PURPOSE: Abacavir, an antiretroviral drug used in HIV therapy associated with myocardial infarction, promotes thrombosis through P2X7 receptors. The role of platelets as pro-thrombotic cells is acknowledged whereas that of neutrophils-due to their secretory capacity-is gaining recognition. This study analyses the role of neutrophils-specifically the secretome of abacavir-treated neutrophils (SNABC )-in platelet activation that precedes thrombosis. EXPERIMENTAL APPROACH: Effects of abacavir or SNABC on platelet activation and platelet-leukocyte interactions and expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) were analysed by flow cytometry. The secretome was analysed by proteomics. The role of leukocytes in the actions of abacavir was evaluated in a mouse model of thrombosis. KEY RESULTS: Abacavir induced platelet-leukocyte interactions, not directly via effects of abacavir on platelets, but via activation of neutrophils, which triggered interactions between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1 (PSGL-1). SNABC stimulated platelet activation and platelet-leukocyte interactions through a process that was dependent on LOX-1, neutrophil P2X7 and platelet P2Y1, P2Y12 and P2X1 receptors. Abacavir induced the expression of LOX-1 on neutrophils and of the soluble form of LOX-1 (sLOX-1) in SNABC . Neutrophils, LOX-1, P2X7, P2Y1, P2Y12 and P2X1 receptors were required for the pro-thrombotic actions of abacavir in vivo. CONCLUSION AND IMPLICATIONS: Neutrophils are target cells in abacavir-induced thrombosis. Abacavir released sLOX-1 from neutrophils via activation of their P2X7 receptors, which in turn activated platelets. Hence, sLOX-1 could be the missing link in the cardiovascular risk associated with abacavir.
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Neutrófilos , Trombosis , Animales , Ratones , Receptores Purinérgicos P2X7/metabolismo , Receptores Depuradores de Clase E , Plaquetas , Trombosis/metabolismo , Selectina-PRESUMEN
Drug-induced liver injury (DILI) constitutes a clinical challenge due to the incomplete characterization of the mechanisms involved and potential risk factors. Efavirenz, an anti-HIV drug, induces deleterious actions in hepatocytes that could underlie induction of the NLRP3 inflammasome, an important regulator of inflammatory responses during liver injury. We assessed the potential of efavirenz to modulate the inflammatory and fibrogenic responses of major liver cell types involved in DILI. The effects of efavirenz were evaluated both in vitro and in vivo. Efavirenz triggered inflammation in hepatocytes, in a process that involved NF-κB and the NLRP3 inflammasome, and activated hepatic stellate cells (HSCs), thereby enhancing expression of inflammatory and fibrogenic markers. The NLRP3 inflammasome was not altered in efavirenz-treated macrophages, but these cells polarized towards the anti-inflammatory M2 phenotype and displayed upregulated anti-inflammatory mediators. Conversely, no evidence of damage was observed in efavirenz-treated animals, except when macrophages were depleted, which resulted in the in vivo manifestation of the deleterious effects detected in hepatocytes and HSCs. Efavirenz elicits a cell-specific activation of the NLRP3 inflammasome in hepatocytes and HSCs, but macrophages appear to counteract efavirenz-induced liver injury. Our results highlight the dynamic nature of the interaction among liver cell populations and emphasize the potential of targeting macrophage polarization as a strategy to treat NLRP3 inflammasome-induced liver injury.
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The coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has resulted in a pandemic with over 270 million confirmed cases and 5.3 million deaths worldwide. In some cases, the infection leads to acute respiratory distress syndrome (ARDS), which is triggered by a cytokine storm and multiple organ failure. Clinical hematological, biochemical, coagulation, and inflammatory markers, such as interleukins, are associated with COVID-19 disease progression. In this regard, neutrophilia, neutrophil-to-lymphocyte ratio (NLR), and neutrophil-to-albumin ratio (NAR), have emerged as promising biomarkers of disease severity and progression. In the pathophysiology of ARDS, the inflammatory environment induces neutrophil influx and activation in the lungs, promoting the release of cytokines, proteases, reactive oxygen species (ROS), and, eventually, neutrophil extracellular traps (NETs). NETs components, such as DNA, histones, myeloperoxidase, and elastase, may exert cytotoxic activity and alveolar damage. Thus, NETs have also been described as potential biomarkers of COVID-19 prognosis. Several studies have demonstrated that NETs are induced in COVID-19 patients, and that the highest levels of NETs are found in critical ones, therefore highlighting a correlation between NETs and severity of the disease. Knowledge of NETs signaling pathways, and the targeting of points of NETs release, could help to develop an effective treatment for COVID-19, and specifically for severe cases, which would help to manage the pandemic.
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The cardiovascular toxicity of Abacavir is related to its purinergic structure. Purinergic P2X7-receptors (P2X7R), characterized by activation by high concentrations of ATP and with high plasticity, seem implicated. We appraise the nature of the interplay between Abacavir and P2X7R in generating vascular inflammation. The effects of Abacavir on leukocyte-endothelium interactions were compared with those of its metabolite carbovir triphosphate (CBV-TP) or ATP in the presence of apyrase (ATP-ase) or A804598 (P2X7R-antagonist). CBV-TP and ATP levels were evaluated by HPLC, while binding of Abacavir, CBV-TP and ATP to P2X7R was assessed by radioligand and docking studies. Hypersensitivity studies explored a potential allosteric action of Abacavir. Clinical concentrations of Abacavir (20 µmol/L) induced leukocyte-endothelial cell interactions by specifically activating P2X7R, but the drug did not show affinity for the P2X7R ATP-binding site (site 1). CBV-TP levels were undetectable in Abacavir-treated cells, while those of ATP were unaltered. The effects of Abacavir were Apyrase-dependent, implying dependence on endogenous ATP. Exogenous ATP induced a profile of proinflammatory actions similar to Abacavir, but was not entirely P2X7R-dependent. Docking calculations suggested ATP-binding to sites 1 and 2, and Abacavir-binding only to allosteric site 2. A combination of concentrations of Abacavir (1 µmol/L) and ATP (0.1 µmol/L) that had no effect when administered separately induced leukocyte-endothelium interactions mediated by P2X7R and involving Connexin43 channels. Therefore, Abacavir acts as a positive allosteric modulator of P2X7R, turning low concentrations of endogenous ATP themselves incapable of stimulating P2X7R into a functional proinflammatory agonist of the receptor.
RESUMEN
There is currently no vaccine available and no specific medication against Coronavirus 2019 disease (COVID-19). The treatment is mainly based on support measures. In this context, several potentially useful therapies have been approved for use in clinical trials, such as convalescent plasma transfusion (CPT). PubMed was searched for studies on convalescent plasma and COVID-19, SARS or MERS. Studies on clinical efficacy in diseases caused by other coronaviruses (SARS-CoV and MERS-CoV) showed clinical improvement, increase of neutralizing antibodies, decreased mortality and absence of adverse events during and after treatment. We found 13 studies on this type of treatment used in patients with severe and critical COVID-19. Despite limitations regarding methodology, number of patients and the protocols for the analysis of donors' convalescent plasma, patients who received CPT showed clinical improvement, improvement of ventilatory patterns, resolution of lung injuries, decreased mortality, improvement of laboratory parameters, increase of neutralizing antibodies, decreased viral load and low frequency of adverse events.
Actualmente no existe vacuna disponible ni medicación específica contra la enfermedad por coronavirus 2019 (COVID-19). El tratamiento se basa fundamentalmente en medidas de soporte. En este contexto, se han aprobado múltiples terapias de potencial utilidad para su uso en ensayos clínicos, como la transfusión de plasma convaleciente (TPC). Se realizó una búsqueda en PubMed de estudios sobre plasma convaleciente y COVID-19, SARS o MERS. Los estudios sobre la eficacia clínica en enfermedades causadas por otros coronavirus (SARS-CoV y MERS-CoV) evidenciaron mejoría clínica, aumento de anticuerpos neutralizantes, disminución de la mortalidad y ausencia de eventos adversos durante y después del tratamiento. En el caso de la COVID-19, se encontraron 13 estudios en pacientes con la COVID-19 grave y crítica. Aunque existen limitaciones en la metodología, en el número de pacientes y en los protocolos para el análisis del plasma convaleciente de los donantes, los pacientes que recibieron TPC evidenciaron mejoría clínica, mejoría de patrones ventilatorios, resolución de lesiones pulmonares, disminución de mortalidad, mejoría de parámetros laboratoriales, aumento de anticuerpos neutralizantes, disminución de carga viral y baja frecuencia de eventos adversos.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , COVID-19/terapia , Transfusión de Componentes Sanguíneos/métodos , COVID-19/inmunología , COVID-19/mortalidad , Humanos , Inmunización Pasiva/efectos adversos , Inmunización Pasiva/métodos , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Carga Viral/inmunología , Sueroterapia para COVID-19RESUMEN
Gastric mucosal inflammation is frequently associated with hypergastrinemia, and a correlation exists between the level of gastrin and degree of gastritis. We have previously observed that gastrin promotes leukocyte-endothelial interactions and contributes to Helicobacter-induced inflammation in the rat mesentery. In the present study, we aimed to evaluate a possible proinflammatory activity of gastrin in humans. The interaction between human leukocytes [U-937 cells, peripheral blood polymorphonuclear (PMN), and peripheral blood mononuclear (PBMC) cells] and human umbilical vein endothelial cells (HUVEC) was analyzed in static and dynamic conditions. The endothelial expression of adhesion molecules [P-selectin, E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule (VCAM)-1] was analyzed by flow cytometry and fluorescent microscopy screening. Gastrin increased the static adhesion of U-937 cells to HUVEC (1 h; 10(-9) M: 122 +/- 9%; 10(-8) M: 143 +/- 17%; 10(-7) M: 162 +/- 14% vs. control, all P < 0.05). Incubation of HUVEC with gastrin (4 h) also increased PBMC rolling (vehicle: 63 +/- 12; 10(-9) M: 109 +/- 29; 10(-8) M: 141 +/- 24; 10(-7) M: 261 +/- 16 leukocytes/min, P < 0.05) and adhesion (vehicle: 3 +/- 2, 10(-9) M: 11 +/- 4, 10(-8) M: 17 +/- 5, 10(-7) M: 15 +/- 5 leukocytes/mm(2), all P < 0.05) in the parallel-plate flow chamber. Treatment of PBMC with gastrin had no effects. The cholecystokinin (CCK)-2 receptor antagonist (L-365,260, 10(-7) M) prevented the effects of gastrin. P-selectin and VCAM-1 expression were enhanced by gastrin, and neutralizing antibodies against these molecules prevented PBMC rolling and adhesion. Gastrin did not affect the interactions between HUVEC and PMN. Gastrin induces interactions between human mononuclear leukocytes and endothelial cells through the activation of CCK-2 receptors and the enhancement of endothelial P-selectin and VCAM-1.
Asunto(s)
Comunicación Celular/fisiología , Células Endoteliales/fisiología , Gastrinas/farmacología , Leucocitos Mononucleares/fisiología , Selectina-P/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Humanos , Rodamiento de Leucocito/efectos de los fármacos , Microscopía Fluorescente , Receptor de Colecistoquinina B/metabolismo , Venas Umbilicales/citologíaRESUMEN
Gastric mucosal inflammation causes hypergastrinemia, and gastrin receptors have been detected in several leukocyte types. We have analyzed whether gastrin affects the leukocyte-endothelial cell interactions in vivo by monitoring leukocyte rolling, adhesion, and emigration in rat mesenteric venules using intravital microscopy. Mesenteric superfusion with exogenous gastrin increased these processes in a concentration- and time-dependent manner, effects prevented by the cholecystokinin (CCK)-2 receptor antagonists (proglumide, L-365,260) but not by the CCK-1 receptor antagonist devazepide. A similar response was induced by exogenous CCK or endogenously released gastrin. CCK-2 receptors were localized in mesenteric macrophages and polymorphonuclear leukocytes. This effect of gastrin is not modulated by somatostatin and is independent of the endogenous release of histamine. To analyze whether hypergastrinemia elicited by Helicobacter pylori (HP) modulates the inflammation induced by the germ, rats were chronically administered with an extract of a CagA+/VacA+ strain of HP. This protocol increased gastrinemia and induced an inflammatory response in the rat mesentery. Blockade of CCK-2 receptors attenuated this response and induced a qualitative change in the leukocyte infiltrate suggestive of a receding inflammatory process. Our results reveal a new proinflammatory role of gastrin that seems to contribute to the maintenance of the inflammation elicited by HP components.