RESUMEN
Trichoderma are fungi that are well-known to inhibit the growth of a variety of plant pathogens. Currently, there is an increasing search for new drugs to treat toxoplasmosis. The aims of this study were to investigate the effect of ExtTs in the control of Toxoplasma gondii proliferation in vitro and the course of toxoplasmosis in a mouse model. Firstly, the cytotoxicity of the ExtTs was evaluated by cultivating macrophages with different concentrations of the extract and cell viability was assessed by the MTT assay. Next, the infectivity of the T. gondii treated with extract was analyzed by infecting J774 macrophages. To evaluate the effect of the ExtTs in vivo, C57BL/6 mice were infected orally with T. gondii, ME-49, treated daily with ExtTs, and clinical, biochemical and histological changes were monitored. It was demonstrated that the extract did not affect the host cellular viability and, the treatment of parasites with ExtTs altered their morphology and decreased their ability to proliferate inside macrophages. Additionally, the treatment of mice with ExtTs decreased the parasitism and inflammation in the small intestine and liver of infected mice in parallel with increased IL-10/TNF ratio systemically and prevented alterations to serum VLDL and triglyceride levels. Thus, ExtTs could be considered an alternative/complementary therapy to control toxoplasmosis.
RESUMEN
Sporotrichosis is a subcutaneous mycosis and is distributed throughout the world, although most cases belong to endemic regions with a warmer climate such as tropical and subtropical areas. The infection occurs mainly by traumatic inoculation of propagules. Similarly, to other organisms, Sporothrix brasiliensis display many biological features that aid in its ability to infect the host, such as extracellular vesicles, bilayered biological structures that provides communication between host cells and between fungi cells themselves. Recently, research on Sporothrix complex have been focused on finding new molecules and components with potential for therapeutic approaches. Here, we study the relationship among EVs and the host's macrophages as well as their role during infection to assess whether these vesicles are helping the fungi or inducing a protective effect on mice during the infection. We found that after cocultivation with different concentrations of purified yeasts EVs from Sb, J774 macrophages displayed an increased fungicidal activity (Phagocytic Index) resulting in lower colony-forming units the more EVs were added, without jeopardizing the viability of the macrophages. Interleukins IL-6, IL-10, and IL-12 were measured during the infection period, showing elevated levels of IL-12 and IL-6 in a dose-dependent manner, but no significant change for IL-10. We also assessed the expression of important molecules in the immune response, such as MHC class II and the immunoglobulin CD86. Both these molecules were overexpressed in Sb yeasts infected mice. Our results indicate that EVs play a protective role during Sporothrix brasiliensis infections.
Asunto(s)
Vesículas Extracelulares , Sporothrix , Esporotricosis , Animales , Macrófagos , RatonesRESUMEN
Oxidized LDL (oxLDL) plays a pivotal role on atherosclerosis development, mainly in the formation of lipid-laden macrophage "foam cells". As a consequence, substances that can modulate LDL oxidation have a pharmacological and therapeutic relevance. Based in previous findings showing the ability of Syzigium cumini leaf extract (ScExt) in preventing LDL oxidation in vitro, this study was aimed to assess the effects of ScExt on oxLDL-mediated toxicity in murine J774 macrophages-like cells. For biochemical analyses, LDL isolated from fresh human plasma and oxidized with CuSO4 was incubated with ScExt pre-treated macrophages. Our results demonstrated that ScExt was efficient in preventing the overproduction of reactive oxygen/nitrogen species (ROS/RNS), the loss of macrophage's viability and the foam cells formation induced by oxLDL. These protective effects of ScExt make it a promising antioxidant for future trials toward atherogenesis.
Asunto(s)
Antioxidantes/farmacología , Aterosclerosis/prevención & control , Macrófagos/efectos de los fármacos , Extractos Vegetales/farmacología , Hojas de la Planta/química , Sustancias Protectoras/farmacología , Syzygium/química , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Espumosas/citología , Células Espumosas/efectos de los fármacos , Humanos , Lipoproteínas LDL/toxicidad , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Antibiotics with new modes of action that are active against intracellular forms of Staphylococcus aureus are sorely needed to fight recalcitrant infections caused by this bacterium. Afabicin desphosphono (Debio 1452, the active form of afabicin [Debio 1450]) is an inhibitor of FabI enoyl-Acyl carrier protein reductase and has specific and extremely potent activity against Staphylococci, including strains resistant to current antistaphylococcal agents. Using mouse J774 macrophages and human THP-1 monocytes, we showed that afabicin desphosphono: (i) accumulates rapidly in cells, reaching stable cellular-to-extracellular concentration ratios of about 30; (ii) is recovered entirely and free in the cell-soluble fraction (no evidence of stable association with proteins or other macromolecules). Afabicin desphosphono caused a maximum cfu decrease of about 2.5 log10 after incubation in broth for 30 h, including against strains resistant to vancomycin, daptomycin, and/or linezolid. Using a pharmacodynamic model of infected THP-1 monocytes (30 h of incubation post-phagocytosis), we showed that afabicin desphosphono is bacteriostatic (maximum cfu decrease: 0.56 to 0.73 log10) towards all strains tested, a behaviour shared with the comparators (vancomycin, daptomycin, and linezolid) when tested against susceptible strains. We conclude that afabicin desphosphono has a similar potential as vancomycin, daptomycin or linezolid to control the intracellular growth and survival of phagocytized S. aureus and remains fully active against strains resistant to these comparators.
Asunto(s)
Antibacterianos/farmacología , Antibacterianos/farmacocinética , Benzofuranos/farmacología , Benzofuranos/farmacocinética , Ácidos Grasos/antagonistas & inhibidores , Naftiridinas/farmacología , Naftiridinas/farmacocinética , Fagocitosis , Staphylococcus aureus/efectos de los fármacos , Animales , Línea Celular , Células Cultivadas , Farmacorresistencia Bacteriana , Ácidos Grasos/biosíntesis , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Modelos BiológicosRESUMEN
Leishmaniasis is a vector borne parasitic disease affecting millions of people worldwide and is spreading into further areas because of global warming. The development of new active substances against these single-cell eukaryotic parasites is of great importance. Leishmania tarentolae promastigotes (LtP) are non-pathogenic for mammals and serve as model organisms for pathogenic Leishmania in basic research. However, it is important to refine methods to study the process of the infection of mammalian macrophages by LtP and pathogenic Leishmania. Important stages of the infection are phagocytosis by macrophages and multiplication of Leishmania amastigotes in the phagolysosome of macrophages. In this study, advanced methods using electron spin resonance (ESR) spectroscopy and genetically manipulated LtP were used to monitor the infection of adherent J774 macrophages with LtP. An ESR method was established to detect the formation of superoxide radicals directly in adherent J774â¯cells and to investigate the effect of LtP on this activity. J774 cells responded with a burst of superoxide radicals in the presence of phorbol myristate acetate as positive control. In contrast, challenging J774â¯cells with LtP resulted in a much lower burst of superoxide radicals. To facilitate LtP detection in the phagolysosome of J774 macrophages, LtP expressing enhanced green fluorescent protein (EGFP-LtP) were constructed. After different infection times with EGFP-LtP, the J774â¯cells were visualized by phase contrast microscopy and the cell number was determined. The intramacrophage Leishmania tarentolae amastigotes (LtA) expressing EGFP were detected by fluorescence microscopy and then counted with ImageJ. These experiments showed that LtP are taken up by J774â¯cells and form intraphagolysosomal amastigotes. LtA under our conditions multiplied intracellularly and were able to persist about 48â¯h in J774â¯cells. These experiments showed that ESR spectroscopy of attached macrophages and the use of the EGFP-LtP are suitable methods to study the initial phase of Leishmania infection in vitro.
Asunto(s)
Leishmania/inmunología , Macrófagos/parasitología , Fagocitosis , Animales , Línea Celular , Espectroscopía de Resonancia por Spin del Electrón , Electroporación , Humanos , Leishmania/genética , Macrófagos/inmunología , Microscopía Fluorescente , Microscopía de Contraste de Fase , Superóxidos/metabolismoRESUMEN
Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5'-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-ß. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-ß-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5'-(α,ß-methylene)diphosphate (APCP; 400µg/paw). Furthermore, we found increased activity of 5'-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A2A receptors.
Asunto(s)
Adenosina/metabolismo , Antiinflamatorios/farmacología , Inhibidores de la Ciclooxigenasa 2/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/sangre , 5'-Nucleotidasa/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Línea Celular , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Dinoprostona/sangre , Edema/tratamiento farmacológico , Edema/inmunología , Edema/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratas Wistar , Receptor de Adenosina A2A/metabolismo , Sulfonamidas/administración & dosificaciónRESUMEN
Chylomicron remnants, which carry dietary fats and cholesterol, play a role in promoting atherosclerosis. Chylomicron remnants are characterized by high cholesterol content at the surface, different from low-density lipoproteins (LDLs) containing high amounts of esterified cholesterol (CE) in the core. We prepared cholesterol-rich emulsions (TO-PC/cholesterol emulsions) as models for chylomicron remnants and compared their effects on J774 macrophages with acetylated-LDL (ac-LDL). Internalization of TO-PC/cholesterol emulsions into macrophages reduced cell viability, whereas ac-LDL did not. Surprisingly, there was no difference in intracellular free cholesterol content between cells incubated with TO-PC/cholesterol emulsions and with ac-LDL. Furthermore, cholesterol in TO-PC/cholesterol emulsions and ac-LDL both were internalized into J774 macrophages; however, incubation with TO-PC/cholesterol emulsions induced leakage of lysosomal protease, cathepsin-L, to cytosol, which was not observed for incubation with ac-LDL. Inhibition of the activity of cathepsin-L recovered the viability of macrophages that ingested TO-PC/cholesterol emulsions. We suggest an alternative fate of cholesterol-rich emulsions taken up by macrophages, which is different from other atherogenic lipoproteins rich in CE; internalization of TO-PC/cholesterol emulsions into macrophages induces rapid free cholesterol accumulation in lysosomes and cell death due to lysosomal destabilization.
Asunto(s)
Colesterol/metabolismo , Remanentes de Quilomicrones/metabolismo , Lisosomas/metabolismo , Macrófagos/metabolismo , Animales , Transporte Biológico , Muerte Celular , Línea Celular , Emulsiones , Lipoproteínas LDL/metabolismo , Macrófagos/patología , Ratones , Fosfatidilcolinas/metabolismo , Trioleína/metabolismoRESUMEN
The present work evaluates the biocompatibility of a fluoride surface-modified AZ31 magnesium alloy (AZ31HF) with different cell lines that coexist in the implant environment to test its potential use as a biodegradable and absorbable biomaterial for bone repair. A clear stimulation of cell proliferation and an enhancement of the mitochondrial respiratory activity were observed when mouse osteoblasts (MC3T3-E1), fibroblasts (L929), and macrophages (J774) cell lines were cultured with the modified alloy. No significant change in apoptosis or viability rates was observed when osteoblasts and fibroblasts cultures were grown in the presence of this alloy. A proteomic analysis of the MC3T3-E1 cell extracts cultured in the presence of AZ31HF showed an overexpression of proteins related with the mineralization process, which is a necessary step for bone repair. An increase in the lactate dehydrogenase activity was observed in the MC3T3-E1 and J774 cell cultures that could be a response of the oxidative stress produced by the presence of the material. This stress could be related to the increase observed in the respiratory mitochondrial activity or respiratory burst measured in theses cultures that indicate damage in the cell membranes and subsequently some cell death. Results reported here, for and against AZ31HF, should be taken into account when considering the potential use of this modified alloy in bone repair applications.