Characterization of Regulatory T Cells in Patients Infected by Leishmania Infantum.

High IL-10 levels are pivotal to parasite survival in visceral leishmaniasis (VL). Antigenic stimuli induce IL-10 expression and release of adenosine by CD39/CD73. Due their intrinsic ability to express IL-10 and produce adenosine from extracellular ATP, we evaluated the IL-10, CD39, and CD73 expression by Regulatory T cells (Treg) correlated with VL pathology. Using flow cytometry, Treg cells was analyzed in peripheral blood samples from VL patients (in the presence and absence of Leishmania infantum soluble antigen (SLA)) and healthy individuals (negative endemic control-NEC group), without any treatment. Additionally, IL-10 levels in leukocytes culture supernatant were measured in all groups by ELISA assay. VL patients presented more Treg frequency than NEC group, independently of stimulation. ELISA results demonstrated that SLA induced higher IL-10 expression in the VL group. However, the NEC group had a higher Treg IL-10+ compared to the VL group without stimulation and SLA restored the IL-10 in Treg. Additionally, an increase in Treg CD73+ in the VL group independently of stimuli compared to that in the NEC group was observed. We suggest that Treg are not the main source of IL-10, while the CD73 pathway may be an attempt to modulate the exacerbation of immune response in VL disease.

Antioxidant and vasorelaxant activities induced by northeastern Brazilian fermented grape skins.

BACKGROUND: In northeastern Brazil, grape pomace has become a potential alternative byproduct because of the recover phenolic compounds from the vinification process. Comparative analyses were performed between lyophilized extract of grape skins from pomace, described as fermented (FGS), and fresh, unfermented (UGS) grape skins to show the relevant brand's composition upon the first maceration in winemaking. METHODS: The use of in vitro testing such as Folin-Ciocalteu's, DPPH free radical scavenger and HPLC methods were performed to evidence antioxidant effect and phenolic compounds. Additionally, vascular reactivity studies were performed in third-order branches of rat superior mesenteric arteries, which were obtained and placed in organ baths containing Krebs-Henseleit solution, maintained at 37 °C, gassed with a mixture of 95% O2 and 5% CO2, and maintained at pH 7.4. The in situ formation of reactive oxygen species (ROS) was evaluated in small mesenteric rings using oxidative fluorescent dihydroethidium dye. RESULTS: We found higher phenolic content and antioxidant activity in FGS when compared to UGS. HPLC analyses identified a significant number of phenolic compounds with antioxidant potential in both samples. The vasorelaxant effect induced by FGS was more potent than that induced by UGS, and the activity was attenuated after removal of vascular endothelium or by blockade of endothelium-derived relaxing factors, such as NO and EDHF. CONCLUSIONS: The FGS extract may be a great source of natural polyphenol products with potent antioxidant effects and endothelium-dependent vasodilatory actions involving NO and EDHF pathways.

Vasorelaxation induced by a new naphthoquinone-oxime is mediated by NO-sGC-cGMP pathway.

It has been established that oximes cause endothelium-independent relaxation in blood vessels. In the present study, the cardiovascular effects of the new oxime 3-hydroxy-4-(hydroxyimino)-2-(3-methylbut-2-enylnaphtalen-1(4H)-one (Oxime S1) derived from lapachol were evaluated. In normotensive rats, administration of Oxime S1 (10, 15, 20 and 30 mg/Kg, i.v.) produced dose-dependent reduction in blood pressure. In isolated aorta and superior mesenteric artery rings, Oxime S1 induced endothelium-independent and concentration-dependent relaxations (10(-8) M to 10(-4) M). In addition, Oxime S1-induced vasorelaxations were attenuated by hydroxocobalamin or methylene blue in aorta and by PTIO or ODQ in mesenteric artery rings, suggesting a role for the nitric oxide (NO) pathway. Additionally, Oxime S1 (30 and 100 µM) significantly increased NO concentrations (13.9 ± 1.6 nM and 17.9 ± 4.1 nM, respectively) measured by nitric oxide microsensors. Furthermore, pre-contraction with KCl (80 mM) prevented Oxime S1-derived vasorelaxation in endothelium-denuded aortic rings. Of note, combined treatment with potassium channel inhibitors also reduced Oxime S1-mediated vasorelaxation suggesting a role for potassium channels, more precisely Kir, Kv and KATP channels. We observed the involvement of BKCa channels in Oxime S1-induced relaxation in mesenteric artery rings. In conclusion, these data suggest that the Oxime S1 induces hypotension and vasorelaxation via NO pathway by activating soluble guanylate cyclase (sGC) and K+ channels.

Nitric oxide as a target for the hypotensive and vasorelaxing effects induced by (Z)-ethyl 12-nitrooxy-octadec-9-enoate in rats.

The cardiovascular effects induced by a new organic nitrate were investigated in rats. The (Z)-ethyl 12-nitrooxy-octadec-9-enoate (NCOE) was synthesized from ricinoleic acid, the major compound of the castor oil. NCOE induced significant and dose-dependent hypotension and bradycardia in normotensive rats. In rats pretreated with NCOE (60 mg/kg, i.v., once a day) for 4 consecutive days, hypotension induced by the nitrate was similar to that observed in rats that were not pretreated with the compound. The vasorelaxation induced by the compound was concentration-dependent (10(-10)-10(-3) M) in rat mesenteric artery rings, pre-contracted with phenylephrine (1 µM), with or without endothelium. Pre-incubation with PTIO (300 µM), a free radical form of NO (NO) scavenger, attenuated the NCOE vasorelaxation potency. However, in the presence of L-cysteine (3 mM), a reduced form of NO (NO-) scavenger, NCOE response was potentiated. NCOE effect was not changed in the presence of an inhibitor of cytochrome P450, proadifen (10 µM). On the other hand, the vasodilation was reduced in the presence of mitochondrial aldehyde dehydrogenase inhibitor (mtALDH), cyanamide (1 mM); soluble guanylyl cyclase inhibitor (sGC), ODQ (10 µM); and non-selective K+ channels blocker, TEA (3 mM). In addition the NCOE-induced vasorelaxation was reduced by BKCa (iberiotoxin, 100 nM) and KATP selective (glibenclamide, 10 µM) blockers, however the effect was not modified by a KV blocker (4-aminopyridine, 1 mM). Furthermore, NCOE increased NO levels in rat aortic smooth muscle cultured cells, detected by NO-sensitive probe DAF-2DA, by flow cytometry. These results together suggest that NCOE induces short-lasting hypotension and bradycardia, and promotes vasorelaxation due to NO release through the compound metabolism via mtALDH and consequent sGC, KATP and BKCa activation. Furthermore, the compound was not able to induce tolerance.

Participation of nitric oxide pathway in the relaxation response induced by E-cinnamaldehyde oxime in superior mesenteric artery isolated from rats.

For many years, nitric oxide (NO) has been studied as an important mediator in the control of vascular tone. Endothelial deficiencies that diminish NO production can result in the development of several future cardiovascular diseases, such as hypertension and arteriosclerosis. In this context, new drugs with potential ability to donate NO have been studied. In this study, 3 aromatic oximes [benzophenone oxime, 4-Cl-benzophenone oxime, and E-cinnamaldehyde oxime (E-CAOx)] induced vasorelaxation in endothelium-denuded and intact superior mesenteric rings precontracted with phenylephrine. E-CAOx demonstrated the most potent effect, and its mechanism of action was evaluated. Vascular reactivity experiments demonstrated that the effect of E-CAOx was reduced by the presence of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one, and (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate, suggesting the participation of NO/sGC/PKG pathway. NO donation seems to be mediated through nicatinamide adenine dinucleotide phosphate-dependent reductases because 7-ethoxyresorufin decreased the effect of E-CAOx on vascular reactivity and reduced NO formation as detected by flow cytometry using the NO indicator diaminofluorescein 4,5-diacetate. Further downstream of NO donation, K+ subtype channels were also shown to be involved in the E-CAOx vasorelaxant effect. The present study showed that E-CAOx acts like an NO donor, activating NO/sGC/PKG pathway and thus K+ channels.

Rotundifolone-induced relaxation is mediated by BK(Ca) channel activation and Ca(v) channel inactivation.

Rotundifolone is the major constituent of the essential oil of Mentha x villosa Hudson. In preliminary studies, rotundifolone induced significant hypotensive, bradycardic and vasorelaxant effects in rats. Thus, to gain more insight into the pharmacology of rotundifolone, the aim of this study was to characterize the molecular mechanism of action involved in relaxation produced by rotundifolone. The relaxant effect was investigated in rat superior mesenteric arteries by using isometric tension measurements and whole-cell patch-clamp techniques. Rotundifolone relaxed phenylephrine-induced contractions in a concentration-dependent manner. Pre-treatment with KCl (20 mM), charybdotoxin (10(-7) M) or tetraethylammonium (TEA 10(-3) or 3 × 10(-3) M) significantly attenuated the relaxation effect induced by rotundifolone. Additionally, whole-cell patch-clamp recordings were made in mesenteric smooth muscle cells and showed that rotundifolone significantly increased K(+) currents, and this effect was abolished by TEA (10(-3) M), suggesting the participation of BK(Ca) channels. Furthermore, rotundifolone inhibited the vasoconstriction induced by CaCl(2) in depolarizing nominally Ca(2+) -free medium and antagonized the contractions elicited by an L-type Ca(2+) channel agonist, S(-)-Bay K 8644 (2 × 10(-7) M), indicating that the vasodilatation involved inhibition of Ca(2+) influx through L-type voltage-dependent calcium channels (Ca(v) type-L). Additionally, rotundifolone inhibited L-type Ca(2+) currents (I(Ca) L), affecting the voltage-dependent activation of I(Ca) L and steady-state inactivation. Our findings suggest that rotundifolone induces vasodilatation through two distinct but complementary mechanisms that clearly depend on the concentration range used. Rotundifolone elicits an increase in the current density of BK(Ca) channels and causes a shift in the steady-state inactivation relationship for Ca(v) type-L towards more hyperpolarized membrane potentials.

Hypotensive and endothelium-independent vasorelaxant effects of methanolic extract from Curcuma longa L. in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma longa L. (CL) is a yellow rhizome that is used in African traditional medicine to treat palpitation, hypertension or other related blood circulation disorders. AIM OF THE STUDY: To justify the use of CL in ethnomedicine, we investigated the vasorelaxant effect of methanolic extract of CL (CLME) and its underlying mechanisms in isolated rat mesenteric artery. MATERIALS AND METHODS: The effect of CLME on the mean arterial pressure (MAP) and heart rate (HR) (pulse interval) were determined in vivo in non-anaesthetized rats. Superior mesenteric rings were isolated, suspended in organ baths containing Tyrode solution at 37 degrees C and gassed with 95% O(2)+5% CO(2), under a resting tension of 0.75 g. The vasorelaxant effects of CLME were studied by means of isometric tension recording experiments. RESULTS: In normotensive rats, CLME (10, 20 and 30 mg/kg, i.v.) induced dose-dependent hypotension (2.0+/-0.5%; 27.1+/-5.0% and 26.7+/-4.6%, respectively), and pronounced bradycardia (5.8+/-1.2%, 19.3+/-3.2% and 22.9+/-4.6%, respectively). CLME (1-1000 microg/mL) induced concentration-dependent relaxation of tonic contractions evoked by phenylephrine (Phe) (10 microM) and KCl (80 mM) in rings with intact-endothelium (E(max)=82.3+/-3.2% and 97.7+/-0.7%) or denuded-endothelium (E(max)=91.4+/-1.0% and 97.8+/-1.1%). Also, in a depolarized, Ca(2+) free medium, CLME inhibited CaCl(2) (1 microM-30 mM)-induced contractions and caused a concentration-dependent rightward shift of the response curves, indicating that CLME inhibited the contractile mechanisms involving extracellular Ca(2+) influx. In addition, in Ca(2+) free media containing EGTA (1 mM), CLME inhibited the transient contraction of denuded rings constricted with Phe, but not those evoked by caffeine (20 mM). In contrast, neither glibenclamide, BaCl(2), tetraethylammonium nor 4-aminopyridine affected CLME-induced relaxation. CONCLUSIONS: These results demonstrate the hypotensive and bradycardic effects of CLME, as well as its potent vasodilation of rat mesenteric arteries. These effects, may in part, be due to the inhibition of extracellular Ca(2+) influx and/or inhibition of intracellular Ca(2+) mobilization from Phe-sensitive stores.