Isolation and chemical analysis of a fatty acid fraction of Kalanchoe pinnata with a potent lymphocyte suppressive activity.

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.

Respiratory effects of lipopolysaccharide-induced inflammatory lung injury in mice.

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.

Immunosuppressive and anti-inflammatory effects of methanolic extract and the polyacetylene isolated from Bidens pilosa L.

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.

Effects of rolipram on cyclic AMP levels in alveolar macrophages and lipopolysaccharide-induced inflammation in mouse lung.

1. Our previous work demonstrated that bacterial lipopolysaccharide (LPS), administered by aerosol, induced tumour necrosis factor (TNF-alpha) synthesis leading to the infiltration of neutrophils into mice lungs. The treatment of animals with prostaglandin E2 or dibutyryl cyclic AMP impaired both processes. In this study, the target cell for LPS and the modulation by cyclic AMP of TNF-alpha production and neutrophil recruitment were investigated. 2. One hour after inhalation of 2 ml of 0.3 mg ml(-1) LPS, TNF-alpha levels measured by an ELISA method increased in the bronchoalveolar lavage fluid (BALF) of BALB/c mice, reaching a maximal level 3 h after inhalation. The immunocytochemistry assay demonstrated that 1 h after inhalation, 21.2% of alveolar macrophages collected in the BALF were immunopositive for TNF-alpha. 3. When mice were pretreated, i.p., with 20 mg kg(-1) rolipram, a selective inhibitor of phosphodiesterase type 4, TNF-alpha levels in the BALF were significantly reduced and only 7.3% of alveolar macrophages were immunopositive for TNF-alpha. 4. Alveolar macrophages from rolipram-treated mice collected 30 min after inhalation of LPS had a significant increase in the intracellular concentrations of cyclic AMP. This was accompanied by a marked reduction of TNF-alpha levels in the BALF that were associated with a suppression of TNF-alpha mRNA expression. 5. Systemic treatment with 20 mg kg(-1) rolipram almost completely inhibited the LPS-induced neutrophil recruitment, whereas it did not significantly reduce the recruitment induced by rmTNF-alpha. 6. Our results indicate that alveolar macrophages may be the target cells for both the induction and control of the lung inflammatory response to LPS. They also suggest that systemic treatment with cyclic AMP-elevating agents may be useful to control local inflammation resulting from inhalation of bacterial endotoxin.

Jatrophone and 12-O-tetradecanoyl phorbol-13-acetate antagonism of stimulation of natural killer activity and lymphocyte proliferation.

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.

Effect of cyclo-oxygenase inhibitors and modulators of cyclic AMP formation on lipopolysaccharide-induced neutrophil infiltration in mouse lung.

1. The adult respiratory distress syndrome (ARDS) is an acute lung inflammation developed after direct or indirect contact with pathogenic agents. In the present study, a mouse model was developed to mimic this condition using aerosolized bacterial lipopolysaccharide (LPS) and to investigate the mechanisms involved in the lung inflammatory response. 2. Inhalation of LPS led to a time and dose-dependent increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the bronchoalveolar lavage fluid (BALF) of Balb/c mice. Under the same conditions, neutrophil infiltration was also found in the BALF of the LPS-sensitive mouse strain C3H/HeN, but was absent in the LPS-resistant strain C3H/HeJ. Intranasal administration of murine recombinant TNF-alpha also triggered neutrophil recruitment. 3. One hour after inhalation of LPS, half of the maximal level of TNF-alpha was measured in the BALF, but only a few neutrophils were detected at this time. The peak TNF-alpha concentration was reached at 3 h, when the neutrophil amount started to increase. At 24 h, maximal neutrophil number was found in the BALF and TNF-alpha was no longer present. 4. Pretreatment of mice under different experimental conditions demonstrated that: (a) cycloheximide almost completely blocks both neutrophil recruitment and TNF-alpha production; (b) anti TNF-alpha antibodies block neutrophil recruitment; (c) indomethacin or aspirin enhance by two fold neutrophil recruitment; (d) indomethacin significantly increases TNF-alpha production 1 h after inhalation of LPS; (e) dibutyryl cyclic AMP and prostaglandin E2 (PGE2) block both neutrophil recruitment and TNF-alpha production. 5. It is concluded that aerosolized LPS in mice triggers an acute lung inflammation which can be used as a potential model of inhalational ARDS and that, strategies leading to the elevation of cyclic AMP levels in vivo can be effective in modulating LPS-induced TNF-alpha synthesis and neutrophil recruitment.

Therapeutic effect of oral Kalanchoe pinnata leaf extract in murine leishmaniasis.

The effect of a leaf extract from Kalanchoe pinnata (Kp) was investigated in BALB/c mice infected with Leishmania amazonensis. Oral treatment with Kp significantly delayed onset of disease as compared to untreated mice or mice receiving Kp by the intravenous or topical routes. When initiated at early stages of infection, daily oral doses of 8 mg prevented lesion growth and the effect was long-lasting, comparable to the reference antileishmanial drug Glucantime. The decreased lesion growth using the oral route was accompanied by a significant decrease in the number of viable parasites. Protection was accompanied by a diminished capacity of animals to develop delayed-type hypersensitivity and to produce specific antibodies.

Patuletin acetylrhamnosides from Kalanchoe brasiliensis as inhibitors of human lymphocyte proliferative activity.

The fractionation of the juice of fresh stems and leaves of Kalanchoe brasiliensis was monitored by an assay measuring lymphocyte proliferative activity and allowed the isolation and identification of seven patuletin rhamnoside derivatives [1-7]. Three of them are novel, namely, patuletin 3-O-(4"-O-acetyl-alpha-L-rhamnopyranosyl)-7-O-(2'"-O-acetyl- alpha-L-rhamnopyranoside) [1], patuletin 3-O-alpha-L-rhamnopyranosyl-7-O-(2'"-O-acetyl-alpha-L- rhamnopyranoside) [3], and patuletin 3-O-(4"-O-acetyl-alpha-L-rhamnopyranosyl)-7-O- rhamnopyranoside [4], and four are known [2, 5-7]. Their structures were determined by the analysis of 1H-1H and 1H-13C COSY nmr, ci, and fab mass spectra.

Inhibition of lymphocyte activation by extracts and fractions of Kalanchoe, Alternanthera, Paullinia and Mikania species.

Aqueous or ethanol extracts obtained from the leaves of Mikania glomerata (Asteraceae) and different species of Kalanchoe (Crassulaceae) or Alternanthera (Amaranthaceae) and from seeds of Paullinia cupana (Sapindaceae) blocked the capacity of human lymphocytes to proliferate in vitro. In all cases, a proliferative response induced by cocultivation of lymphocytes with phytohemagglutinin was inhibited even after depletion of phagocytic mononuclear cells by adherence. A flavonoid-enriched fraction from K. pinnata and an ethyl acetate fraction from A. tenella were 10 to 20-fold, respectively, more potent in inhibiting lymphocyte proliferation than their original crude extracts. In contrast, these fractions had no inhibitory action on human natural killer activity. Lymphocyte suppression by the extracts was not due to a cytotoxic effect. Pre-incubation with the highest extract concentrations did not affect the capacity of lymphocytes to proliferate, after removal of extracts from the medium. These results indicate a direct action of the extracts on lymphocytes. They also suggest that these medicinal plants may contain potential immunosuppressive substances that selectively act on activation steps of the cells of the immune system.

Opposite effects of amiloride and amiloride analogues on activation of natural killer cytotoxicity by the phorbol ester TPA and gamma-interferon.

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.

Antivenom and biological effects of ar-turmerone isolated from Curcuma longa (Zingiberaceae)

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.

Dimethyl sulfoxide: a possible effect on the interconversion of phosphorylated forms of Na+,K(+)-ATPase.

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.

Lack of sensitivity to ouabain in natural killer activity.

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.

ATP synthesis by the (Na+ + K+)-ATPase in the absence of an ionic gradient. Effects of organic solvent.

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.

Exchange between inorganic phosphate and adenosine triphosphate in (Na+,K+)-ATPase.

(Na+,K+)-ATPase is able to catalyze a continuous ATP in equilibrium Pi exchange in the presence of Na+ and in the absence of a transmembrane ionic gradient. At pH 7.6 the Na+ concentration required for half-maximal activity is 85 mM and at pH 5.1 it is 340 mM. In the presence of optimal Na+ concentration, the rate of exchange is maximal at pH 6.0 and varies with ADP and Pi concentration in the assay medium. ATP in equilibrium Pi exchange is inhibited by K+ and by ouabain.

Evidence of competitive mechanisms during oxidation of CS(2)N(-)(3) by permanganate ion in alkaline solution.

The oxidation of the 1,2,3,4-thiatriazole-5-thiolate ion, CS(2)N(-)(3), by permanganate ions in alkaline medium has been investigated over a wide range of oxidant concentrations. With a moderate excess of permanganate ion a 17-electron reaction takes place, yielding sulphate, nitrogen and carbon dioxide. With a high permanganate concentration the pseudohalide undergoes a 26-electron reaction yielding sulphate, carbon dioxide and nitrite as final products. These two different stoichiometries arise from the existence of competitive mechanisms and the ratio C(MnO(-)(4))/C(CS(2)N(-)(3)) will determine the course of the reaction.