Quercetin ameliorates experimental autoimmune myocarditis in rats.

PURPOSE: Experimental autoimmune myocarditis (EAM) in rats is an animal model of human giant cell myocarditis and post-myocarditis dilated cardiomyopathy. The pathogenesis of EAM has not been elucidated, but there is accumulating evidence that cytokines secreted from monocytes/macrophages and T cells play a crucial role in the induction and progression of disease. Flavonoids are a large group of polyphenolic compounds abundantly present in the human diet, which scavenge oxygen radicals and have anti-inflammatory activities. Having in mind in vivo beneficial effects of flavonoid quercetin in different animal models of immunoinflammatory diseases such as experimental autoimmune encephalomyelitis and adjuvant arthritis, on the one side, and its in vitro suppressive effect on production of tumor necrosis factor-alpha (TNF-α) on the other side, we investigated the effects of quercetin on EAM in rats. METHODS: Myocarditis was induced in Dark Agouti (DA) rats by injection of porcine cardiac myosin and quercetin at doses of 10 or 20 mg/kg was orally administered from days 0 to 21 after induction of disease. The severity of myocarditis was evaluated by determination of heart weight/body weight ratio (Hw/Bw) and histopathological examination of hearts. The levels of cytokines (TNF-α, IL-12, IL-17 and IL-10) in serum and lymph node cells (LNC) culture supernatants were measured by ELISA. RESULTS: The rats treated with 20 mg/kg of quercetin had significantly decreased incidence of EAM, Hw/Bw, macroscopic and microscopic scores of hearts. Further, in EAM rats treated with quercetin levels of TNF-α and IL-17 were significantly lower, while the level of IL-10 was significantly higher both in serum and culture supernatants of LNC stimulated with concanavalin A compared with vehicle-treated animals. CONCLUSIONS: The present study suggests that quercetin ameliorates EAM, at least in part, by interfering production of proinflammatory (TNF-α and IL-17) and/or anti-inflammatory (IL-10) cytokines.

Insulin mimetic effect of tungsten compounds on isolated rat adipocytes.

Investigations of effective, orally active, and safe antidiabetic metallopharmaceuticals have been carried out during the last two decades. It has been reported that tungsten compounds mimic the action of insulin in intact cell systems. As insulin mimetics, the most investigated tungsten compound was sodium tungstate (ST), rarely investigated was tungstophosphoric acid (WPA), but never alanine complex of tungstophosphoric acid (WPA-A). In this study, the insulin mimetic activity of three different tungsten compounds, ST, WPA, and WPA-A, was evaluated by means of in vitro measurements of the glucose uptake and inhibition of free fatty acids release from epinephrine-treated isolated rat white adipocytes. We investigated the influence of concentration (lower and higher, 0.1 and 1.0 mM, respectively) and solvent: isotonic salt solution-saline (0.9% w/v of NaCl) and dimethyl sulfoxide (DMSO; 2% v/v), on the biological effect of tested compounds. Our experimental data showed that all of the three investigated tungsten compounds possess insulin mimetic activity in vitro on the isolated adipocytes. Influence of concentration and solvents on insulin mimetic effect for the certain tungsten compounds were: WPA was shown effect independently of concentration and solvents; higher concentration and DMSO were significant decreasing insulin mimetic effect of ST; lower concentration and saline led to decreasing effect of WPA-A. Generally, there were no differences in insulin mimetic effect of three tungsten compounds in lower concentration and dissolved in DMSO. When saline was used as solvent, it was needed higher concentration of investigated compounds to accomplish the same effect. In conclusion, our results suggest that low concentration (0.1 mM) of ST, WPA, and WPA-A dissolved in 2% DMSO could be the good candidates for in vivo investigation of their antidiabetic properties.

Comparative effects of aspirin and NO-releasing aspirins on differentiation, maturation and function of human monocyte-derived dendritic cells in vitro.

Acetylsalicilyc acid (aspirin, ASA) is a well known anti-inflammatory drug with immunomodulatory properties. NO-releasing aspirins (NO-ASA) are new compounds with anti-inflammatory properties. We studied the effects of ASA and two NO-ASA (NCX 4016 and NCX 4040) on human monocyte-derived dendritic cells (MoDC). Immature MoDC were generated in vitro from monocytes in the presence of recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. Mature MoDC were obtained by adding lipopolysaccharide (LPS) in cultures of immature MoDC. As we found that ASA at 4-8 mM, NCX 4016 at 400-800 microM and NCX 4040 at 4-8 microM stimulated apoptosis of monocytes and immature MoDC, sub-apoptotic concentrations of ASA (2 mM), NCX 4016 (200 microM) and NCX 4040 (2 microM) were used in experiments. Examined substances were added at the beginning of MoDC cultivation. MoDC differentiated in the presence of examined compounds had lower expression of HLA-DR, CD80, CD40 and CD54, decreased allostimulatory activity and lower production of IL-12 p40. ASA and NCX 4016 decreased production of IL-10, whereas NCX 4040 had the opposite effect. ASA inhibited the expression of CD1a and prevented downregulation of CD14, NCX 4016 stimulated the differentiation of CD1a+CD14+ and CD1a(-)CD14+ cells, whereas NCX 4040 decreased the proportion of CD1a+CD14(-) and increased the frequency of CD1a+CD14+ cells, compared to control. Maturation, both in ASA and NO-ASA treated MoDC was characterized by decreased allostimulatory activity, lower expression of CD83, HLA-DR, costimulatory molecules and CD54 and decreased production of IL-10 and IL-12 p40. In conclusion, we confirmed that ASA impairs differentiation, maturation and function of MoDC and found that NCX 4016 and NCX 4040 exerted similar, but not identical effects at about 10- and 1000-fold lower concentrations, respectively, compared to ASA.

Beneficial effects of dimethyl fumarate on experimental autoimmune myocarditis.

BACKGROUND: Fumaric acid esters (FAE) have been proven to be effective for the systemic treatment of psoriasis and multiple sclerosis, Th1 cell-mediated chronic inflammatory diseases, but their effect on autoimmune myocarditis has not yet been addressed. We investigated the effect of dimethyl fumarate (DMF) on myosin-induced experimental autoimmune myocarditis (EAM). METHODS: Dark Agouti (DA) rats immunized with porcine cardiac myosin were orally treated with 5 and 15 mg/kg body weight (bw) DMF either from days 0-10 (early treatment groups) or from days 10-21 (late treatment groups) after induction of EAM. All rats were sacrificed on day 21 after immunization and hearts were evaluated macroscopically and microscopically. Levels of TNF-alpha and IL-10 in serum and lymph node cells culture supernatants were detected by ELISA. RESULTS: Both early and late treatment with 15 mg/kg body weight (bw) DMF markedly reduced the severity of myocarditis by comparing the incidence, heart weight/bw ratio, macroscopic and microscopic scores, and number of OX-6+ cells in the myocardium. Further, levels of tumor necrosis factor-alpha (TNF-alpha) in serum and culture supernatants of lymph node cells stimulated with ConA or myosin were significantly lower in DMF-treated EAM animals compared with vehicle-treated EAM rats. There was no significant difference in serum levels of interleukin-10 between DMF- and vehicle-treated EAM rats. CONCLUSIONS: These results show for the first time that DMF ameliorates experimental autoimmune myocarditis and may be acted, at least in part, by interfering with the production of TNF-alpha.

Antimicrobial, anti-inflammatory, anti-ulcer and antioxidant activities of Carlina acanthifolia root essential oil.

The root of Carlina acanthifolia All. (Asteraceae) has been traditionally used in the treatment of various disorders including stomach and skin diseases. We studied antimicrobial, anti-inflammatory, anti-ulcer and antioxidant activities of Carlina acanthifolia root essential oil, in order to validate some of the ethnopharmacological claims. Antimicrobial activity was tested on 15 bacteria and three strains of fungi using the agar diffusion and broth microdilution methods. In assessing anti-inflammatory activity the carrageenan-induced rat paw oedema test was used, while ethanol-induced stress gastric ulcer test in rats was used in testing anti-ulcer activity. Antioxidant properties were evaluated trough the effect of the essential oil on lipid peroxidation (TBA assay) and its capability of quenching 2,2-diphenyl-1-picrylhydrazyl (DPPH) and OH radicals. The oil expressed significant antimicrobial activity, being the most active against Gram (+) bacteria: Streptococcus pyogenes, Enterococcus faecalis, Bacillus subtilis and against Candida albicans. In all applied concentrations, Carlina acanthifolia root essential oil reduced carrageenan-induced rat paw oedema in dose-dependent manner, achieving high degree of anti-inflammatory activity. The effect was comparable with that of indomethacin used as a reference drug. In the ethanol-induced stress gastric ulcer test in rats, it was shown that the tested essential oil produced significant dose-dependent gastroprotective activity. The results also pointed out substantial and dose-dependent antioxidant activity of the investigated essential oil, with carlina oxide as the main antioxidant component.

Comparison of two different protocols for the induction of maturation of human dendritic cells in vitro.

BACKGROUND: Dendritic cells (DC) have been used for immunotherapy of malignant tumors, different kinds of infections, and other clinical conditions. For that purpose, optimal conditions for the generation of functionally mature DC in vitro are required. Two different protocols for the induction of maturation of monocyte-derived DC (MDDC) were compared in this study. METHODS: MDDC were generated in vitro by cultivating adherent monocytes of healthy volunteers with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) during 6-days period. The immature DC thus prepared were induced to mature using two protocols. DC were stimulated for 2 days with lipopolysaccharide (LPS), or with a cocktail of proinflammatory mediators (PM) containing IL-1beta, IL-6, tumor necrosis factor alpha (TNFalpha), and prostaglandin E2 (PGE2), respectively. Phenotypic characteristics of MDDC and their endocytic activity were studied by flow cytometry. Allostimulatory activity of these cells was tested in the mixed leukocyte reaction (MLR), whereas the production of cytokines was determined by ELISA kits. RESULTS: MDDC matured with PM (PM-DC) were predominantly non-adherent cells, while about 30% of LPS-matured DC were adherent cells. In comparison with LPS-DC, PM-DC expressed higher levels of CD86 and CD83, had lower endocytic activity, produced higher levels of IL-10 and lower levels of IL-12, and more strongly stimulated proliferation of allogeneic lymphocytes. CONCLUSION: The protocol based on the combination of proinflammatory cytokines and PGE2 is better for the induction of maturation of human MDDC in vitro than the protocol using LPS alone.