RESUMEN
Previously we demonstrated that Kalanchoe pinnata (KP) leaf extracts inhibited in vitro lymphocyte proliferation and showed in vivo immunosuppressive activity. Here we attempt to identify the immunosuppressive substances present in KP guided by the lymphoproliferative assays. From the ethanolic extract was purified a fraction (KP12SA) twenty-fold more potent to block murine lymphocyte proliferation than the crude extract. Chemical analysis by 1H- and 13C-NMR, IR and GC-MS of KP12SA (methylated sample) showed 89.3% of palmitic acid (C16), 10.7% of stearic acid (C18) and traces of arachidic (C20) and behenic acids (C22). This study provides evidence that fatty acids present in Kalanchoe pinnata may be responsible, at least in part, for its immunosuppressive effect in vivo.
Asunto(s)
Ácidos Grasos/farmacología , Inmunosupresores/farmacología , Extractos Vegetales/farmacología , Plantas Medicinales/química , Linfocitos T/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Ácidos Grasos/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Linfocitos T/citologíaRESUMEN
The pathogenic mechanisms of lipopolysaccharide (LPS)-induced lung injury have not been classified. This study examined the physiological changes after endotoxin inhalation and related those to features of pulmonary inflammation in mice. Pulmonary mechanics, histopathology, and bronchoalveolar lavage fluid (BALF) from BALB/c mice were analysed at different occasions (3, 24, 48 and 72 h) after inhalation of saline or LPS from Escherichia coli (0.3 (L0.3) or 10 mg x mL(-1) (L10)). Mice were sedated, anaesthetized, and ventilated. After chest wall resection static (Est) and dynamic (Edyn) elastances, deltaE (Edyn-Est), resistive (deltaP1) and viscoelastic/inhomogeneous pressures (deltaP2), and deltaP1+deltaP2 (deltaPtot) were obtained by end-inflation occlusion method. Lungs were prepared for histopathology. In parallel groups, tumour necrosis factor (TNF)-alpha, neutrophils, and protein were evaluated in the BALF. L0.3 and L10 showed a time-dependent production of TNF-alpha preceding a massive neutrophil infiltration. In L10 BALF there was an increase in protein level at 24 and 48 h. Est and Edyn increased early in L0.3 (65%, 63%) and L10 (41%, 51%). In L10 deltaE, deltaP2, and deltaPtot showed a gradual rise. At 72 h all groups were similar. L0.3 showed an early increase in cellularity, which returned to normal at 72 h. L10 presented the same pattern with the cell count remaining elevated until 72 h. In conclusion, lipopolysaccharide inhalation led to elastic and viscoelastic pulmonary changes together with tumour necrosis factor-alpha production and neutrophil infiltration in mouse lung.
Asunto(s)
Escherichia coli/inmunología , Lipopolisacáridos/inmunología , Neumonía Bacteriana/inmunología , Mecánica Respiratoria/inmunología , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Infiltración Neutrófila/inmunología , Neumonía Bacteriana/patología , Edema Pulmonar/inmunología , Edema Pulmonar/patología , Pruebas de Función Respiratoria , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The immunomodulatory effect of the methanolic extract obtained from dried leaves of Bidens pilosa L. (Asteraceae) and the polyacetylene 2-O-beta-D-glucosyltrideca-11 E-en-3,5,7,9-tetrayn-1,2-diol (PA-1) isolated from it was investigated. The extract inhibited the proliferative response in two in vitro models: human lymphocytes stimulated by 5 microg ml(-1) phytohemagglutinin (PHA) or to 100 nM 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus 0.15 microM ionomycin and murine lymphocytes stimulated by 5 microg ml(-1) concanavalin A (Con A) or in the mixed leukocyte reaction (IC50 = 12.5 to 25 microg ml(-1)). PA-1 was 10-told more potent than the original extract in blocking both human and murine lymphocyte proliferation (IC50 = 1.25 to 2.5 microg ml(-1)). In mice, the intraperitoneal (i.p.) administration of methanolic extract of B. pilosa significantly reduced the size of the popliteal lymph node (PLN) after the inflammation induced by zymosan. One week after the injection of zymosan (150 microg) in the foot pad, PLN weighed 4.6 +/- 0.6 mg in comparison with 0.5 +/- 0.07 mg of the contralateral non-inflamed foot pad. The i.p. treatment with 10 mg extract from day 2 to day 6 after zymosan injection reduced the PLN weight to 1.8 +/- 0.3 mg. The data suggest an immunosuppressive activity of components of B. pilosa that may explain its popularly perceived anti-inflammatory effect.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inmunosupresores/farmacología , Extractos Vegetales/farmacología , Acetileno/análogos & derivados , Acetileno/aislamiento & purificación , Acetileno/farmacología , Animales , Antiinflamatorios no Esteroideos/aislamiento & purificación , Asteraceae , Humanos , Inmunosupresores/aislamiento & purificación , Técnicas In Vitro , Inflamación/tratamiento farmacológico , Inflamación/patología , Activación de Linfocitos/efectos de los fármacos , Metanol , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Extractos Vegetales/aislamiento & purificación , Polímeros/aislamiento & purificación , Polímeros/farmacología , PoliinosRESUMEN
We have recently reported that the diterpene jatrophone antagonizes the effects of phorbol ester in pharmacogical studies. In order to investigate further whether this action is associated with an inhibition of protein kinase C activity, we examined the effect of jatrophone on the stimulation of lymphocyte activities which are dependent on the protein kinase C pathway. Jatrophone (0.02-0.32 microM) caused concentration-dependent and equipotent inhibition of human lymphocyte proliferation induced by 5 micrograms/ml of phytohemagglutinin or by a combination of 100 ng/ml of 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus 0.15 microM ionomicyn, with IC50 values (and their 95% confidence limits) of 53.4 (42.6-65.3) nM and 48.4 (39.4-59.8) nM, respectively. Jatrophone also blocked, in a concentration-dependent fashion, the murine lymphocyte proliferation stimulated by 5 micrograms/ml of concanavalin A, with an IC50 value of 63.5 (51.2-76.5) nM. The inhibition was not due to a toxic effect as the pre-incubation of lymphocytes for 48 h with 0.32 microM jatrophone did not impair the proliferation after removal of the diterpene from the culture medium. Human lymphocytes when pre-treated with 10 ng/ml TPA had a 3 times higher spontaneous natural killer activity against K562 cells and an increased expression of CD69. In addition, jatrophone inhibited both spontaneous and TPA-stimulated natural killer activity and the expression of CD69. Jatrophone concentrations that inhibited 75% of lymphocyte proliferation did not impair the intracellular increase in Ca2+ flux in lymphocytes stimulated by phytohemagglutinin. These results indicate that jatrophone is a potent inhibitor of activation of lymphocytes, probably through inhibition of the protein kinase C pathway.
Asunto(s)
División Celular/efectos de los fármacos , Diterpenos/farmacología , Células Asesinas Naturales/efectos de los fármacos , Linfocitos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Calcio/metabolismo , Humanos , Lectinas Tipo C , Linfocitos/citología , Linfocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Fitohemaglutininas/farmacologíaRESUMEN
Amiloride, a K(+)-sparing diuretic used as an Na+/H+ exchange inhibitor, blocked the activation of human natural killer (NK) activity against K562 cells by either the phorbol ester TPA or gamma-interferon. However, this stimulation was not blocked by 5-(N,N-hexamethylene)amiloride, a potent inhibitor of Na+/H+ exchange. Spontaneous NK activity was inhibited by this amiloride analogue as well as by 5-(N-methyl-N-guanidinocarbonylmethyl) amiloride, another potent inhibitor of exchange, but only in concentrations 30-80 times higher than those used to inhibit Na+/H+ exchange. The analogue phenamil amiloride blocked NK activity in concentrations found to inhibit epithelial Na+ channels, whereas tetrodotoxin, the specific inhibitor of voltage-dependent Na+ channels, had no effect. These results indicate that Na+/H+ exchange is not essential either for spontaneous NK activity or for its activation by TPA and gamma-interferon. They also suggest the involvement of voltage-independent Na+ channels in NK activity.
Asunto(s)
Amilorida/análogos & derivados , Amilorida/farmacología , Interferón gamma/farmacología , Células Asesinas Naturales/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Canales de Sodio/inmunología , Tetrodotoxina/farmacologíaRESUMEN
A potent antivenom against snakebite was isolated from Curcuma longa, a plant commonly used in traditional Brazilian medicine. The fraction consisting of ar-turmerone neutralized both the hemorrhagic activity present in Bothrops jararaca venom, and the lethal effect of Crotalus durissus terrificus venom in mice. Immunological studies demonstrated that this fraction also inhibited the proliferation and the natural killer activity of human lymphocytes.
Asunto(s)
Cetonas/química , Plantas Medicinales/química , Venenos de Serpiente/antagonistas & inhibidores , Tolueno/análogos & derivados , Animales , Brasil , Supervivencia Celular/efectos de los fármacos , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/toxicidad , Hemorragia/inducido químicamente , Hemorragia/patología , Hemorragia/prevención & control , Humanos , Técnicas In Vitro , Cetonas/farmacología , Células Asesinas Naturales/efectos de los fármacos , Dosificación Letal Mediana , Linfocitos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Ratones , Extractos Vegetales/farmacología , Sesquiterpenos , Venenos de Serpiente/toxicidad , Tolueno/química , Tolueno/farmacologíaRESUMEN
Purified Na+,K(+)-ATPase from kidney outer medulla was phosphorylated by Pi in a reaction synergistically stimulated by Mg2+, when 40% (v/v) dimethyl sulfoxide was added to the assay medium. The phosphoenzyme formed at this solvent concentration was able to synthesize ATP even in the presence of Mg2+, because hydrolysis was impaired. ATP in equilibrium [32P]Pi exchange was also inhibited, indicating that partial reactions in the forward direction were blocked by the solvent. In 40% (v/v) dimethyl sulfoxide the enzyme's affinity for ADP decreased, in comparison with the values observed in purely aqueous medium. Addition of K+, which accelerated dephosphorylation of Na+,K(+)-ATPase in a totally water medium, partially reversed the inhibition of hydrolysis that was observed in the presence of dimethyl sulfoxide.
Asunto(s)
Dimetilsulfóxido/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Hexoquinasa/farmacología , Concentración de Iones de Hidrógeno , Riñón/enzimología , Magnesio/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Potasio/farmacología , Ovinos , AguaRESUMEN
Natural killer (NK) activity against K562 target cells is not an ouabain-sensitive process. Inhibition of 40% of cytotoxicity was achieved only with an ouabain concentration much higher than that required to inhibit cell activation in other systems such as leukocyte chemotaxis and B lymphocyte plaque formation. Pretreatment of effector cells with biological agents such as phorbol-ester 12-O-tetradecanoylphorbol-13-acetate or interferon increased the cytotoxicity. This activation was not counteracted by ouabain. The effect of ouabain on NK activity was compared with a well-known ouabain-sensitive process, for example, phytohemagglutinin-induced peripheral blood lymphocyte proliferation. Ouabain completely blocked [3H]thymidine incorporation, independent of the stage of the culture when the drug was added, with exception of the last 6 h. This inhibition could be partially reversed by addition of KCl. Ouabain was equally effective when whole blood cultures were used. These results suggest that NK activity is ouabain resistant, unlike other systems of cell activation that lead or do not lead to proliferation.
Asunto(s)
Células Asesinas Naturales/inmunología , Linfocitos/fisiología , Ouabaína/farmacología , División Celular/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Humanos , Interferones/farmacología , Células Asesinas Naturales/efectos de los fármacos , Potasio/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
A 200-fold decrease in the Pi concentration required for half-maximal phosphorylation of the (Na+ + K+)-ATPase is observed when 40% (v/v) dimethyl sulfoxide is added to the assay medium. The phosphoenzyme formed in the presence of dimethyl sulfoxide is able to transfer its phosphate to to form ATP when concentrations of organic solvent and of Na+, and is inhibited by ouabain.