Tamoxifen as a potential antileishmanial agent: efficacy in the treatment of Leishmania braziliensis and Leishmania chagasi infections

The aim of this study was to evaluate the efficacy of tamoxifen in vivo in experimental models of cutaneous (CL) and visceral leishmaniasis (VL) caused by Leishmania braziliensis and Leishmania chagasi, respectively. Drug activity was assessed against intracellular amastigotes by treating infected macrophage cultures and evaluating the number of infected cells. In vivo efficacy of tamoxifen was tested in L. braziliensis-infected BALB/c mice and in L. chagasi-infected hamsters. Treatment with 20 mg/kg/day tamoxifen was administered for 15 days by the intraperitoneal route. Efficacy was evaluated through measurements of lesion size, parasite burden at the lesion site or liver and spleen and survival rate. Tamoxifen killed L. braziliensis and L. chagasi intracellular amastigotes with 50% inhibitory concentrations (IC(50)) of 1.9 +/- 0.2 and 2.4 +/- 0.3 mu M, respectively. Treatment of L. braziliensis-infected mice with tamoxifen resulted in significant reductions in lesion size and 99% decrease in parasite burden, compared with mock-treated controls. L. chagasi-infected hamsters treated with tamoxifen showed significant reductions in liver parasite load expressed as Leishman-Donovan units and 95% to 98% reduction in spleen parasite burden. All animals treated with tamoxifen survived while 100% of the mock-treated animals had died by 11 weeks after the interruption of treatment. Tamoxifen is effective in the treatment of CL and VL in rodent models.

Reduced allergic lung inflammation in rats following formaldehyde exposure: Long-term effects on multiple effector systems

Clinical and experimental evidences show that formaldehyde (FA) exposure has an irritant effect on the upper airways. As being an indoor and outdoor pollutant, FA is known to be a causal factor of occupational asthma. This study aimed to investigate the repercussion of FA exposure on the course of a lung allergic process triggered by an antigen unrelated to FA. For this purpose, male Wistar rats were subjected to FA inhalation for 3 consecutive days (1%, 90-min daily), subsequently sensitized with ovalbumin (OVA)-alum via the intraperitoneal route, and 2 weeks later challenged with aerosolized OVA. The OVA challenge in rats after FA inhalation (FA/OVA group) evoked a low-intensity lung inflammation as indicated by the reduced enumerated number of inflammatory cells in bronchoalveolar lavage as compared to FA-untreated allergic rats (OVA/OVA group). Treatment with FA also reduced the number of bone marrow cells and blood leukocytes in sensitized animals challenged with OVA, which suggests that the effects of FA had not been only localized to the airways. As indicated by passive cutaneous anaphylactic reaction, FA treatment did not impair the anti-OVA IgE synthesis, but reduced the magnitude of OVA challenge-induced mast cell degranulation. Moreover, FA treatment was associated to a diminished lung expression of PECAM-1 (platelet-endothelial cell adhesion molecule 1) in lung endothelial cells after OVA challenge and an exacerbated release of nitrites by BAL-cultured cells. Keeping in mind that rats subjected solely to either FA or OVA challenge were able to significantly increase the cell influx into lung, our study shows that FA inhalation triggers long-lasting effects that affect multiple mediator systems associated to OVA-induced allergic lung such as the reduction of mast cells activation, PECAM-1 expression and exacerbation of NO generation, thereby contributing to the decrease of cell recruitment after the OVA challenge. In conclusion, repeated expositions to air-borne FA may impair the lung cell recruitment after an allergic stimulus, thereby leading to a non-responsive condition against inflammatory stimuli likely those where mast cells are involved. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

In Vitro Activity of the Antifungal Plant Defensin RsAFP2 against Candida Isolates and Its In Vivo Efficacy in Prophylactic Murine Models of Candidiasis

We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.

Fenofibrate and Pioglitazone Do Not Ameliorate the Altered Vascular Reactivity in Aorta of Isoproterenol-treated Rats

Chronic stimulation of beta-adrenoceptors with isoproterenol induces alteration of vascular reactivity and increases local proinflammatory cytokines. We investigated whether fenofibrate and pioglitazone, PPAR-alpha and -gamma agonists, respectively, improve the changes in vascular reactivity induced by isoproterenol. Wistar rats received isoproterenol (0.3 mg.kg(-1).day(-1), SC) or vehicle (CT) plus fenofibrate (alpha, 100 mg.kg(-1).day(-1), PO), pioglitazone (gamma, 2.5 mg.kg(-1).day(-1), PO), or water for 7 days. In aortas, isoproterenol treatment enhanced the maximal response (Rmax) to phenylephrine (10(-10) to 10(-4) M) compared to CT as previously demonstrated. The effects of endothelium removal (E-) or L-NAME incubation (100 mu M) on the phenylephrine response were smaller in isoproterenol-treated animals compared to CT while superoxide dismutase (SOD, 150 U/mL) significantly reduced the Rmax to phenylephrine to CT levels. Neither fenofibrate nor pioglitazone changed the effects induced by isoproterenol in aorta. E-, L-NAME, or SOD effects were similar between CT alpha and CT. However, pioglitazone per se increased Rmax to phenylephrine (CT: 59 +/- 4 versus CT gamma: 72 +/- 5 % of contraction to KCl). E- or L-NAME effects were reduced in CT gamma compared to CT, and SOD normalized the altered reactivity to phenylephrine in the CT gamma group. In conclusion, neither fenofibrate nor pioglitazone ameliorates the altered vascular reactivity present in aorta from isoproterenol-treated rats. Moreover, pioglitazone per se induced endothelial dysfunction and increased phenylephrine-induced contraction in aorta.

Perinatal salt restriction: A new pathway to programming adiposity indices in adult female Wistar rats

Low birth weight has been associated with increased obesity in adulthood. It has been shown that dietary salt restriction during intrauterine life induces low birth weight and insulin resistance in adult Wistar rats. The present study had a two-fold objective: to evaluate the effects that low salt intake during pregnancy and lactation has on the amount and distribution of adipose tissue; and to determine whether the phenotypic changes in fat mass in this model are associated with alterations in the activity of the renin-angiotensin system. Maternal salt restriction was found to reduce birth weight in male and female offspring. In adulthood, the female offspring of dams fed the low-salt diet presented higher adiposity indices than those seen in the offspring of dams fed a normal-salt diet. This was attributed to the fact that adipose tissue mass (retroperitoneal but not gonadal, mesenteric or inguinal) was greater in those rats than in the offspring of dams fed a normal diet. The adult offspring of dams fed the low-salt diet, compared to those dams fed a normal-salt diet, presented the following: plasma leptin levels higher in males and lower in females; plasma renin activity higher in males but not in females; and no differences in body weight, mean arterial blood pressure or serum angiotensin-converting enzyme activity. Therefore, low salt intake during pregnancy might lead to the programming of obesity in adult female offspring. (c) 2009 Elsevier Inc. All rights reserved.

Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin

Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund`s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated. (C) 2011 Elsevier Ltd. All rights reserved.

TLR2 and TLR4 expression after kidney ischemia and reperfusion injury in mice treated with FTY720

Ischemia and reperfusion injury (IR) is an antigen independent inflammatory process that causes tissue damage. After IR, kidneys up-regulate leukocyte adhesion molecules and toll-like receptors (TLRs). Moreover, injured kidneys can also secrete factors (i.e. heat shock protein) which bind to TLRs and trigger intracellular events culminating with the increase in the gene expression of inflammatory cytokines. FTY720 is an immunomodulatory compound and protects at least in part kidneys submitted to IR. The mechanisms associated with FTY720`s beneficial effects on kidneys after IR remain elusive. We investigated whether FTY720 administration in mice submitted to kidney IR is associated with modulation of TLR2 and TLR4 expression. C57BL/6 mice submitted to 30 min of renal pedicles clamp were evaluated for serum parameters (creatinine, urea and nitric oxide), kidney histology, spleen and kidney infiltrating cells expression of TLR2 and TLR4, resident kidney cells expression of TLR2 and TLR4 and IL-6 protein expression in kidney. FTY720-treated mice presented decrease in serum creatinine, urea and nitric oxide, diminished expression of TLR2 and TLR4 both in spleen and kidney infiltrating cells, and reduced kidney IL-6 protein expression in comparison with IR non-treated mice. However, acute tubular necrosis was present both in IR non-treated and IR + FTY720-treated groups. Also, FTY720 did not prevent TLR2 and TLR4 expression in kidney resident cells. In conclusion, FTY720 can promote kidney function recovery after IR by reducing the inflammatory process. Further studies are needed in order to establish whether TLR2 and TLR4 down regulation should be therapeutically addressed as protective targets of renal function and structure after IR. (C) 2011 Elsevier B.V. All rights reserved.

Time-dependent increases in ouabain-sensitive Na(+), K(+)-ATPase activity in aortas from diabetic rats: The role of prostanoids and protein kinase C

Aims: Na(+), K(+)-ATPase activity contributes to the regulation of vascular contractility and it has been suggested that vascular Na(+), K(+)-ATPase activity may be altered during the progression of diabetes; however the mechanisms involved in the altered Na(+), K(+)-ATPase activity changes remain unclear. Thus, the aim of the present study was to evaluate ouabain-sensitive Na(+), K(+)-ATPase activity and the mechanism(s) responsible for any alterations on this activity in aortas from 1- and 4-week streptozotocin-pretreated (50 mg kg(-1), i.v.) rats. Main methods: Aortic rings were used to evaluate the relaxation induced by KCl (1-10 mM) in the presence and absence of ouabain (0.1 mmol/L) as an index of ouabain-sensitive Na(+), K(+)-ATPase activity. Protein expression of COX-2 and p-PKC-beta II in aortas were also investigated. Key findings: Ouabain-sensitive Na(+), K(+)-ATPase activity was unaltered following 1-week of streptozotocin administration, but was increased in the 4-week diabetic aorta (27%). Endothelium removal or nitric oxide synthase inhibition with L-NAME decreased ouabain-sensitive Na(+), K(+)-ATPase activity only in control aortas. In denuded aortic rings, indomethacin. NS-398, ridogrel or Go-6976 normalized ouabain-sensitive Na(+), K(+)-ATPase activity in 4-week diabetic rats. In addition, COX-2 (51%) and p-PKC-beta II (59%) protein expression were increased in 4-week diabetic aortas compared to controls. Significance: In conclusion, diabetes led to a time-dependent increase in ouabain-sensitive Na(+), K(+)-ATPase activity. The main mechanism involved in this activation is the release of TxA(2)/PGH(2) by COX-2 in smooth muscle cells, linked to activation of the PKC pathway. (C) 2010 Elsevier Inc. All rights reserved.

Insulin temporal sensitivity and its signaling pathway in the rat pineal gland

Aims: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. Main methods: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3: AANAT complex, respectively. Key findings: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced ""night"". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IR beta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. Significance: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland. (C) 2010 Elsevier Inc. All rights reserved.

Bradykinin inducible receptor is essential to lipopolysaccharide-induced acute lung injury in mice

Lipopolysaccharides from gram-negative bacteria are amongst the most common causative agents of acute lung injury, which is characterized by an inflammatory response, with cellular infiltration and the release of mediators/cytokines. There is evidence that bradykinin plays a role in lung inflammation in asthma but in other types of lung inflammation its role is less clear. In the present study we evaluated the role of the bradykinin B(1) receptor in acute lung injury caused by lipopolysaccharide inhalation and the mechanisms behind bradykinin actions participating in the inflammatory response. We found that in C57BI/6 mice, the bradykinin B(1) receptor expression was up-regulated 24 h after lipopolysaccharide inhalation. At this time, the number of cells and protein concentration were significantly increased in the bronchoalveolar lavage fluid and the mice developed airway hyperreactivity to methacholine. In addition, there was an increased expression of tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma and chemokines (monocytes chemotactic protein-1 and KC) in the bronchoalveolar lavage fluid and in the lung tissue. We then treated the mice with a bradykinin B, receptor antagonist, R-954 (Ac-Orn-[Oic(2), alpha-MePhe(5), D-beta Nal(7), Ile(8)]desArg(9)-bradykinin), 30 min after lipopolysaccharide administration. We observed that this treatment prevented the airway hyperreactivity as well as the increased cellular infiltration and protein content in the bronchoalveolar lavage fluid. Moreover, R-954 inhibited the expression of cytokines/chemokines. These results implicate bradykinin, acting through B(1) receptor, in the development of acute lung injury caused by lipopolysaccharide inhalation. (C) 2010 Elsevier B.V. All rights reserved.

MicroRNA expression is differentially altered by xenobiotic drugs in different human cell lines

Several noncoding microRNAs (miR or miRNA) have been shown to regulate the expression of drug-metabolizing enzymes and transporters. Xenobiotic drug-induced changes in enzyme and transporter expression may be associated with the alteration of miRNA expression. Therefore, this study investigated the impact of 19 xenobiotic drugs (e. g. dexamethasone, vinblastine, bilobalide and cocaine) on the expression of ten miRNAs (miR-18a, -27a, -27b, -124a, -148a, -324-3p, -328, -451, -519c and -1291) in MCF-7, Caco-2, SH-SY5Y and BE(2)-M17 cell systems. The data revealed that miRNAs were differentially expressed in human cell lines and the change in miRNA expression was dependent on the drug, as well as the type of cells investigated. Notably, treatment with bilobalide led to a 10-fold increase of miR-27a and a 2-fold decrease of miR-148a in Caco-2 cells, but no change of miR-27a and a 2-fold increase of miR-148a in MCF-7 cells. Neuronal miR-124a was generally down-regulated by psychoactive drugs (e. g. cocaine, methadone and fluoxetine) in BE(2)-M17 and SH-SY5Y cells. Dexamethasone and vinblastine, inducers of drug-metabolizing enzymes and transporters, suppressed the expression of miR-27b, -148a and -451 that down-regulate the enzymes and transporters. These findings should provide increased understanding of the altered gene expression underlying drug disposition, multidrug resistance, drug-drug interactions and neuroplasticity. Copyright (C) 2011 John Wiley & Sons, Ltd.

DNA damage induced by the anthracycline cosmomycin D in DNA repair-deficient cells

Anthracyclines have been widely used as antitumor agents, playing a crucial role in the successful treatment of many types of cancer, despite some side effects related to cardiotoxicity. New anthracyclines have been designed and tested, but the first ones discovered, doxorubicin and daunorubicin, continue to be the drugs of choice. Despite their extensive use in chemotherapy, little is known about the DNA repair mechanisms involved in the removal of lesions caused by anthracyclines. The anthracycline cosmomycin D is the main product isolated from Streptomyces olindensis, characterized by a peculiar pattern of glycosylation with two trisaccharide rings attached to the A ring of the tetrahydrotetracene. We assessed the induction of apoptosis (Sub-G(1)) by cosmomycin D in nucleotide excision repair-deficient fibroblasts (XP-A and XP-C) as well as the levels of DNA damage (alkaline comet assay). Treatment of XP-A and XP-C cells with cosmomycin D resulted in apoptosis in a time-dependent manner, with highest apoptosis levels observed 96 h after treatment. The effects of cosmomycin D were equivalent to those obtained with doxorubicin. The broad caspase inhibitor Z-VAD-FMK strongly inhibited apoptosis in these cells, and DNA damage induced by cosmomycin D was confirmed by alkaline comet assay. Cosmomycin D induced time-dependent apoptosis in nucleotide excision repair-deficient fibroblasts. Despite similar apoptosis levels, cosmomycin D caused considerably lower levels of DNA damage compared to doxorubicin. This may be related to differences in structure between cosmomycin D and doxorubicin.

Involvement of proteinase-activated receptors 1 and 2 in spreading and phagocytosis by murine adherent peritoneal cells: Modulation by the C-terminal of S100A9 protein

Proteinase-activated receptors (PAR) are widely recognized for their modulatory properties in inflammatory and immune responses; however, their direct role on phagocyte effector functions remains unknown. S100A9, a protein secreted during inflammatory responses, deactivates activated peritoneal macrophages, and its C-terminal portion inhibits spreading and phagocytosis of adherent peritoneal cells. Herein, the effect of PAR1 and PAR2 agonists was investigated on spreading and phagocytosis by adherent peritoneal cells, as well as the ability of murine C-terminal of S100A9 peptide (mS100A9p) to modulate this effect. Adherent peritoneal cells obtained from mouse abdominal cavity were incubated with PAR1 and PAR2 agonists and spreading and phagocytosis of Candida albicans particles were evaluated. PAR1 agonists increased both the spreading and the phagocytic activity, but PAR2 agonists only increased the spreading index. mS100A9p reverted both the increased spreading and phagocytosis induced by PAR1 agonists, but no interference in the increased spreading induced by PAR2 agonists was noticed. The shorter homologue peptide to the C-terminal of mS100A9p, corresponding to the H(92)-E(97) region, also reverted the increased spreading and phagocytosis induced by PAR1 agonists. These findings show that proteinase-activated receptors have an important role for spreading and phagocytosis of adherent peritoneal cells, and that the pepticle corresponding to the C-terminal of S100A9 protein is a remarkable candidate for use as a novel compound to modulate PAR1 function. (C) 2009 Elsevier B.V. All rights reserved.

Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes

Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics. (C) 2009 Elsevier Inc. All rights reserved.

EFFECT OF EXERCISE ON GLUTAMINE SYNTHESIS AND TRANSPORT IN SKELETAL MUSCLE FROM RATS

P>Reductions in plasma glutamine are observed after prolonged exercise. Three hypotheses can explain such a decrease: (i) high demand by the liver and kidney; (ii) impaired release from muscles; and (iii) decreased synthesis in skeletal muscle. The present study investigated the effects of exercise on glutamine synthesis and transport in rat skeletal muscle. Rats were divided into three groups: (i) sedentary (SED; n = 12); (ii) rats killed 1 h after the last exercise bout (EX-1; n = 15); and (iii) rats killed 24 h after the last exercise bout (EX-24; n = 15). Rats in the trained groups swam 1 h/day, 5 days/week for 6 weeks with a load equivalent to 5.5% of their bodyweight. Plasma glutamine and insulin were lower and corticosterone was higher in EX-1 compared with SED rats (P < 0.05 and P < 0.01, respectively). Twenty-four hours after exercise (EX-24), plasma glutamine was restored to levels seen in SED rats, whereas insulin levels were higher (P < 0.001) and costicosterone levels were lower (P < 0.01) than in EX-1. In the soleus, ammonia levels were lower in EX-1 than in SED rats (P < 0.001). After 24 h, glutamine, glutamate and ammonia levels were lower in EX-24 than in SED and EX-1 rats (P < 0.001). Soleus glutamine synthetase (GS) activity was increased in EX-1 and was decreased in EX-24 compared with SED rats (both P < 0.001). The decrease in plasma glutamine concentration in EX-1 is not mediated by GS or glutamine transport in skeletal muscle. However, 24 h after exercise, lower GS may contribute to the decrease in glutamine concentration in muscle.