Chronic methionine load-induced hyperhomocysteinemia impairs the relaxation induced by bradykinin in the isolated rat carotid

This study investigates the effects of chronic methionine intake on bradykinin (BK)-relaxation. Vascular reactivity experiments were performed on carotid rings from male Wistar rats. Treatment with methionine (0.1, 1 or 2 g kg(-1) per day) for 8 and 16 weeks, but not for 2 and 4 weeks, reduced the relaxation induced by BK. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and SQ29548, a selective thromboxane A(2) (TXA(2))/prostaglandin H(2) (PGH(2)) receptor antagonist prevented the reduction in BK-relaxation observed in the carotid from methionine-treated rats. Conversely, AH6809, a selective prostaglandin F(2 alpha) (PGF(2 alpha)) receptor antagonist did not alter BK-relaxation in the carotid from methionine-treated rats. The nitric oxide synthase (NOS) inhibitors L-NAME, L-NNA and 7-nitroindazole reduced the relaxation induced by BK in carotids from control and methionine-treated rats. In summary, we found that chronic methionine intake impairs the endothelium-dependent relaxation induced by BK and this effect is due to an increased production of endothelial vasoconstrictor prostanoids (possibly TXA(2)) that counteracts the relaxant action displayed by the peptide.

Fluorescent indication that nitric oxide formation in NTS neurons is modulated by glutamate and GABA

Nitric oxide (NO) in NTS plays an important role in regulating autonomic function to the cardiovascular system. Using the fluorescent dye DAF-2 DA, we evaluated the NO concentration in NTS. Brainstem slices of rats were loaded with DAF-2 DA, washed, fixed in paraformaldehyde and examined under fluorescent light. In different experimental groups, NTS slices were pre-incubated with 1 mM L-NAME (a non-selective NOS inhibitor), 1 MM D-NAME (an inactive enantiomere of L-NAME), 1 mM kynurenic acid (a nonselective ionotropic receptors antagonist) or 20 mu M bicuculline (a selective GABA(A) receptors antagonist) before and during DAF-2 DA loading. Images were acquired using a confocal microscope and the intensity of fluorescence was quantified in three antero-posterior NTS regions. In addition, slices previously loaded with DAF-2 DA were incubated with NeuN or GFAP antibody. A semi-quantitative analysis of the fluorescence intensity showed that the basal NO concentration was similar in all antero-posterior aspects of the NTS (rostral intermediate, 15.5 +/- 0.8 AU: caudal intermediate, 13.2 +/- 1.4 AU; caudal commissural, 13.8 +/- 1.4 AU, n = 10). In addition, the inhibition of NOS and the antagonism of glutamatergic receptors decreased the NO fluorescence in the NTS. On the other hand, D-NAME did not affect the NO fluorescence and the antagonism of GABAA receptors increased the NO fluorescence in the NTS. It is important to note that the fluorescence for NO was detected mainly in neurons. These data show that the fluorescence observed after NTS loading with DAF-2 DA is a result of NO present in the NTS and support the concept that NTS neurons have basal NO production which is modulated by L-glutamate and GABA. (C) 2009 Elsevier Inc. All rights reserved.

Suramin inhibits macrophage activation by human group IIA phospholipase A(2), but does not affect bactericidal activity of the enzyme

Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A(2) (hsPLA(2)GIIA). The hsPLA(2)GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA(2) GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. The hydrolytic activity of the hsPLA(2) GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA(2) GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA(2) GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 mu M. These results demonstrate that suramin selectively inhibits the activity of the hsPLA(2) GIIA against macrophages, whilst leaving the anti-bacterial function unchanged.

Cyclic Guanosine Monophosphate Signaling Cascade Mediates Pigment Aggregation in Freshwater Shrimp Chromatophores

The cell signaling cascades that mediate pigment movements in crustacean chromatophores are not yet well established, although Ca(2+) and cyclic nucleotide second messengers are involved. Here, we examine the participation of cyclic guanosine monophosphate (cGMP) in pigment aggregation triggered by red pigment concentrating hormone (RPCH) in the red ovarian chromatophores of freshwater shrimp. In Ca(2+)-containing (5.5 mmol l(-1)) saline, 10 mu mol l(-1) dibutyryl cGMP alone produced complete pigment aggregation with the same time course (approximate to 20 min) and peak velocity (approximate to 17 mu m/min) as 10(-8) mol l(-1) RPCH; however, in Ca(2+)-free saline (9 X 10(-11) mol l(-1) Ca(2+)), db-cGMP was without effect. The soluble guanylyl cyclase (GC-S) activators sodium nitroprusside (SNP, 0.5 mu mol l(-1)) and 3-morpholinosydnonimine (SIN-1, 100 mu mol l(-1)) induced moderate aggregation by themselves (approximate to 35%-40%) but did not affect RPCH-triggered aggregation. The GC-S inhibitors zinc protoporphyrin IX (ZnPP-XI, 30 mu mol l(-1)) and 6-anilino-5,8-quinolinedione (LY83583, 10 mu mol l(-1)) partially inhibited RPCH-triggered aggregation by approximate to 35%. Escherichia coli heat-stable enterotoxin (STa, 1 mu mol l(-1)), a membrane-receptor guanylyl cyclase stimulator, did not induce or affect RPCH-triggered aggregation. We propose that the binding of RPCH to an unknown membrane-receptor type activates a Ca(2+)-dependent signaling cascade coupled via cytosolic guanylyl cyclase and cGMP to protein kinase G-phosphorylated proteins that regulate aggregation-associated, cytoskeletal molecular motor activity. This is a further example of a cGMP signaling cascade mediating the effect of a crustacean X-organ neurosecretory peptide.

Therapeutic administration of KM+ lectin protects mice against Paracoccidioides brasiliensis infection via interleukin-12 production in a Toll-like receptor 2-dependent mechanism

KM+ is a mannose-binding lectin from Artocarpus integrifolia that induces interleukin (IL)-12 production by macrophages and protective T helper I immune response against Leishmania major infection. in this study, we performed experiments to evaluate the therapeutic activity of jackfruit KM+ (jfKM(+)) and its recombinant counterpart (rKM(+)) in experimental paracoccidioidomycosis. To this end, jfKM(+) or rKM(+) was administered to BALB/c mice 10 days after infection with Paracoccidiodes brasiliensis. Thirty days postinfection, lungs from the KM+-treated mice contained significantly fewer colony-forming units and little to no organized granulomas compared to the controls. In addition, lung homogenates from the KM+-treated mice presented higher levels of nitric oxide, IL-12, interferon-gamma, and tumor necrosis factor-a, whereas higher levels of IL-4 and IL-10 were detected in the control group. With mice deficient in IL-12, Toll-like receptor (TLR) 2, TLR4, or TLR adaptor molecule MyD88, we demonstrated that KM+ led to protection against P. brasiliensis infection through IL-12 production, which was dependent on TLR2. These results demonstrated a beneficial effect of KM+ on the severity of P. brasiliensis infection and may expand its potential use as a novel immunotherapeutic molecule.

Antioxidant Activity of Uruguayan Propolis. In Vitro and Cellular Assays

The antioxidant capacity of propolis from the southern region of Uruguay was evaluated using in vitro as well as cellular assays. Free radical scavenging capacity was assessed by ORAC, obtaining values significantly higher than those of other natural products (8000 mu mol Trolox equiv/g propolis). ORAC values correlated well with total polyphenol content (determined by Folin-Ciocalteu method) and UV absorption. Total polyphenol content (150 mg gallic acid equiv/g propolis) and flavonoids (45 mg quercetin equiv/g propolis) were similar to values reported for southern Brazilian (group 3) and Argentinean propolis. Flavonoid composition determined by RP-HPLC indicates a strong poplar-tree origin. Samples high in polyphenols efficiently inhibit low-density lipoprotein lipoperoxidation and tyrosine nitration. In addition, Uruguayan propolis was found to induce the expression of endothelial nitric oxide synthase and inhibit endothelial NADPH oxidase, suggesting a potential cardiovascular benefit by increasing nitric oxide bioavailability in the endothelium.

Local immunological factors associated with recurrence of mucosal leishmaniasis

Recurrence of mucosal leishmaniasis (ML) is frequent, but the causative mechanisms are unknown. Our aim was to compare cellular and cytokine patterns of lesions from ML that evolved to recurrence or cure in order to determine the risk factor associated with recurrence. Lesions were evaluated by immunohistochemistry before and after therapy, and patients were followed-up for five years. Higher levels of CD4(+) T and IFN-gamma-producing cells were detected in active lesions and decreased after therapy. Macrophages and IL-10 were markedly increased in cured patients. Conversely, CD8(+) T and NK cells were higher in relapsed than in cured cases. Notably, a decrease in these cells in addition to decreased IL-10 and IFN-gamma was also observed after therapy. These data suggest that exacerbated CD8(+) activity, in addition to a poor regulatory response, could underlie an unfavorable fate with regard to ML. These markers may be useful for predicting the prognosis of ML in lesion studies. (C) 2008 Elsevier Inc. All rights reserved.

Effects of antenatal corticosteroid treatment on pulmonary ventilation and circulation in neonatal lambs with hypoplastic lungs

Our aim was to determine whether antenatal corticosteroids improve perinatal adaptation of the pulmonary circulation in lambs with lung hypoplasia (LH). LH was induced in 12 ovine fetuses between 105 and 140 days gestation (term similar to 147 days); in 6 of these the ewe was given a single dose of betamethasone (11.4 mg im) 24 hr before delivery (LH + B). All lambs, including a control group (n = 6), were delivered at similar to 140 days and ventilated for 2 hr during which arterial pressures, pulmonary blood flow (PBF), and ventilating pressure and flow were recorded. During ventilation, respiratory system compliance was lower in both LH + B and LH groups than in controls. Pulmonary vascular resistance (PVR) was lower in LH + B lambs than in LH lambs and similar to controls; PBF was reduced in LH lambs but was restored to control levels by betamethasone. The mean density of small arteries of LH + B lambs was similar to that of LH lambs (P = 0.06) and lower than in controls; the thickness of the media of small pulmonary arteries from LH + B lambs was similar to that in LH lambs and thicker than in controls. VEGF mRNA levels were not different between groups. PDGF mRNA levels in LH + B lambs were higher than in LH lambs; a similar trend (P = 0.06) was seen for PECAM-1. SP-C mRNA levels were greater in both LH and LH + B lambs than in controls. Effects of betamethasone were greater on indices of pulmonary circulation than ventilation. We conclude that a single dose of maternal betamethasone 24 hr prior to birth has significant favorable effects on the postnatal adaptation of the pulmonary circulation in lambs with LH.

Effects of chronic L-NAME treatment lung tissue mechanics, eosinophilic and extracellular matrix responses induced by chronic pulmonary inflammation

The importance of lung tissue in asthma pathophysiology has been recently recognized. Although nitric oxide mediates smooth muscle tonus control in airways, its effects on lung tissue responsiveness have not been investigated previously. We hypothesized that chronic nitric oxide synthase (NOS) inhibition by N-omega-nitro-L-arginine methyl ester (L-NAME) may modulate lung tissue mechanics and eosinophil and extracellular matrix remodeling in guinea pigs with chronic pulmonary inflammation. Animals were submitted to seven saline or ovalbumin exposures with increasing doses (1 similar to 5 mg/ml for 4 wk) and treated or not with L-NAME in drinking water. After the seventh inhalation (72 h), animals were anesthetized and exsanguinated, and oscillatory mechanics of lung tissue strips were performed in baseline condition and after ovalbumin challenge (0.1%). Using morphometry, we assessed the density of eosinophils, neuronal NOS (nNOS)- and inducible NOS (iNOS)-positive distal lung cells, smooth muscle cells, as well as collagen and elastic fibers in lung tissue. Ovalbumin-exposed animals had an increase in baseline and maximal tissue resistance and elastance, eosinophil density, nNOS- and iNOS-positive cells, the amount of collagen and elastic fibers, and isoprostane-8-PGF(2 alpha) expression in the alveolar septa compared with controls (P < 0.05). L-NAME treatment in ovalbumin-exposed animals attenuated lung tissue mechanical responses (P < 0.01), nNOS- and iNOS-positive cells, elastic fiber content (P < 0.001), and isoprostane-8-PGF(2 alpha) in the alveolar septa (P < 0.001). However, this treatment did not affect the total number of eosinophils and collagen deposition. These data suggest that NO contributes to distal lung parenchyma constriction and to elastic fiber deposition in this model. One possibility may be related to the effects of NO activating the oxidative stress pathway.

Exhaled nitric oxide for monitoring childhood asthma inflammation compared to sputum analysis, serum interleukins and pulmonary function

The level of fractional exhaled nitric oxide (FENO) is significantly elevated in uncontrolled asthma and decreases after anti-inflammatory therapy The aim of this prospective study was to analyze the behavior of FENO in the follow-up and management of the inflammation in asthmatic pediatric patients treated with inhaled corticosteroids (ICS), compared to sputum cellularity, serum interleukins (IL), and pulmonary function. Twenty-six clinically stable asthmatic children aged from 6 to 18 years, previously treated or not with ICS were included. Following an international consensus (GINA), the patients were submitted to standard treatment with inhaled fluticasone for 3 months according to the severity of the disease. During this period, each patient underwent three assessments at intervals of approximately 6 weeks: Each evaluation consisted of the measurement of FENO, determination of serum interleukins IL-5, IL-10, IL-13, and interferon gamma (INF-gamma), spirometry and cytological analysis of spontaneous or induced sputum. A significant reduction in mean FENO and IL-5, without concomitant changes in FEV1, was observed along the study. There was no significant correlation between FeNO and FEV1 in the three assessments. A significant correlation between FeNO and IL-5 levels was only observed in the third assessment (r = 0.499, P=0.025). In most patients, serum IL-10, IL-13, and INF-gamma concentrations were undetectable throughout the study Sputum samples were obtained spontaneously in 11 occasions and in 56 by induction with 3% hypertonic saline solution (success rate: 50.8%), with 39 (69.9%) of them adequate for analysis. Only two of the 26 patients produced adequate samples in the three consecutive evaluations, which impaired the determination of a potential association between sputum cellularity and FeNO levels throughout the study. In conclusion, among the parameters of this study, it was difficult to perform and to interpret the serial analysis of spontaneous or induced sputum. Serum interleukins, which remained at very low or undetectable levels in most patients, were not found to be useful for therapeutic monitoring, except for IL-5 that seems to present some correlation with levels of FeNO exhaled. Monitoring of the mean FEV1 indicated no significant variations during the treatment, demonstrating that functional stability or the absence of obstruction may not reflect the adequate management of asthma. Serial measurement of FeNO seemed to best reflect the progressive anti-inflammatory action of ICS in asthma.

The time course of vasoconstriction and endothelin receptor A expression in pulmonary arterioles of mice continuously exposed to ambient urban levels of air pollution

The present study aimed to verify the time course of the effects of environmental levels of urban air pollution toxicity on lung arterioles. BALB/c mice (n = 56) were continuously exposed to selective chambers equipped with (filtered, F) or without (non-filtered, NF) filter devices for particles and toxic gases for 24 h/day, over 14, 21, 30 or 45 days. After exposure, we evaluated the lumen-wall relationship (an estimator of arteriolar narrowing), endothelial nitric oxide synthase (eNOS) and endothelin type A receptor (ETAr) expression in the vascular wall and inflammatory influx of the peribronchiolar area. Concentrations of fine particulate matter (PM <= 2.5 mu g/m(3)), nitrogen dioxide (NO(2)), black smoke (BS), humidity and temperature in both the environment and inside the chambers were measured daily. Filters cleared 100% of BS and 97% of PM inside the F chamber. The arteriole wall of the lungs of mice from NF chamber had an increased ETAr expression (p <= 0.042) concomitant to a decrease in the lumen/wall ratio (p = 0.02) on the early days of exposure, compared to controls. They also presented a progressive increment of inflammatory influx in the peribronchiolar area during the study (p = 0.04) and decrement of the eNOS expression on the 45th day of exposure in both vascular layers (p <= 0.03). We found that after 14 days of exposure, the ambient levels of air pollutants in Sao Paulo induced vasoconstriction that was associated with an increase in ETAr expression. These vascular results do not appear to be coupled to the progressive inflammatory influx in lung tissue, suggesting a down-regulation of vasoconstrictive mechanisms through an imbalance in the cytokines network. It is likely that these responses are protective measures that decrease tissue damage brought about by continuous exposure to air pollutants. (C) 2010 Elsevier Inc. All rights reserved.

Effects of Exercise Training in Patients with Chronic Heart Failure and Sleep Apnea

Study Objectives: To test the effects of exercise training on sleep and neurovascular control in patients with systolic heart failure with and without sleep disordered breathing. Design: Prospective interventional study. Setting: Cardiac rehabilitation and exercise physiology unit and sleep laboratory. Patients: Twenty-five patients with heart failure, aged 42 to 70 years, and New York Heart Association Functional Class I-III were divided into 1 of 3 groups: obstructive sleep apnea (n = 8), central sleep apnea (n 9) and no sleep apnea (n = 7). Interventions: Four months of no-training (control) followed by 4 months of an exercise training program (three 60-minute, supervised, exercise sessions per week). Measures and Results: Sleep (polysomnography), microneurography, forearm blood flow (plethysmography), peak VO(2). and quality of life were evaluated at baseline and at the end of the control and trained periods. No significant changes occurred in the control period. Exercise training reduced muscle sympathetic nerve activity (P < 0.001) and increased forearm blood flow (P < 0.01), peak VO(2) (P < 0.01), and quality of life (P < 0.01) in all groups, independent of the presence of sleep apnea. Exercise training improved the apnea-hypopnea index, minimum O(2) saturation, and amount stage 3-4 sleep (P < 0.05) in patients with obstructive sleep apnea but had no significant effects in patients with central sleep apnea. Conclusions. The beneficial effects of exercise training on neurovascular function, functional capacity, and quality of life in patients with systolic dysfunction and heart failure occurs independently of sleep disordered breathing. Exercise training lessens the severity of obstructive sleep apnea but does not affect central sleep apnea in patients with heart failure and sleep disordered breathing.

Expression of vitamin D receptor mRNA in the hippocampal formation of rats submitted to a model of temporal lobe epilepsy induced by pilocarpine

Vitamin D (VD), is a steroid hormone with multiple functions in the central nervous system (CNS), producing numerous physiological effects mediated by its receptor (VDR). Clinical and experimental studies have shown a link between VD dysfunction and epilepsy. Along these lines, the purpose of our work was to analyze the relative expression of VDR mRNA in the hippocampal formation of rats during the three periods of pilocarpine-induced epilepsy. Male Wistar rats were divided into five groups: (1) control group; rats that received saline 0.9%, i.p. and were killed 7 days after its administration (CTRL, n = 8), (2) SE group; rats that received pilocarpine and were killed 4 h after SE (SE, n = 8), (3) Silent group-7 days; rats that received pilocarpine and were killed 7 days after SE (SIL 7d, n = 8), (4) Silent group-14 days; rats that received pilocarpine and were killed 14 days after SE (SIL 14d, n = 8), (5) Chronic group; rats that received pilocarpine and were killed 60 days after the first spontaneous seizure, (chronic, n = 8). The relative expression of VDR mRNA was determined by real-time PCR. Our results showed an increase of the relative expression of VDR mRNA in the SIL 7 days, SIL 14 days and Chronic groups, respectively (0.060 +/- 0.024; 0.052 +/- 0.035; 0.085 +/- 0.055) when compared with the CTRL and SE groups (0.019 +/- 0.017; 0.019 +/- 0.025). These data suggest the VDR as a possible candidate participating in the epileptogenesis process of the pilocarpine model of epilepsy. (C) 2008 Elsevier Inc. All rights reserved.

Effects of bilateral adrenalectomy on systemic kainate-induced activation of the nucleus of the solitary tract. Regulation of blood pressure and local neurotransmitters

Glutamatergic transmission through metabotropic and ionotropic receptors, including kainate receptors, plays an important role in the nucleus of the solitary tract (NTS) functions. Glutamate system may interact with several other neurotransmitter systems which might also be influenced by steroid hormones. In the present study we analyzed the ability of systemic kainate to stimulate rat NTS neurons, which was evaluated by c-Fos as a marker of neuronal activation, and also to change the levels of NTS neurotransmitters such as GABA, NPY, CGRP, GAL, NT and NO by means of quantitative immunohistichemistry combined with image analysis. The analysis was also performed in adrenalectomized and kainate stimulated rats in order to evaluate a possible role of adrenal hormones on NTS neurotransmission. Male Wistar rats (3 month-old) were used in the present study. A group of 15 rats was submitted either to bilateral adrenalectomy or sham operation. Forty-eight hours after the surgeries, adrenalectomized rats received a single intraperitoneal injection of kainate (12 mg/kg) and the sham-operated rats were injected either with saline or kainate and sacrificed 8 hours later. The same experimental design was applied in a group of rats in order to register the arterial blood pressure. Systemic kainate decreased the basal values of mean arterial blood pressure (35%) and heart rate (22%) of sham-operated rats, reduction that were maintained in adrenalectomized rats. Kainate triggered a marked elevation of c-Fos positive neurons in the NTS which was 54% counteracted by adrenalectomy. The kainate activated NTS showed changes in the immunoreactive levels of GABA (143% of elevation) and NPY (36% of decrease), which were not modified by previous ablation of adrenal glands. Modulation in the levels of CGRP, GAL and NT immunoreactivities were only observed after kainate in the adrenalectomized rats. Treatments did not alter NOS labeling. It is possible that modulatory function among neurotransmitter systems in the NTS might be influenced by steroid hormones and the implications for central regulation of blood pressure or other visceral regulatory mechanisms control should be further investigated.

Immunohistochemical evaluation of macrophage activity and its relationship with apoptotic cell death in the polar forms of leprosy

The objective of the present study was to investigate the correlation between macrophage activity and apoptosis in the polar forms of leprosy because the immunopathological phenomena involved in these forms are still poorly understood For this purpose, 29 skin biopsy samples obtained from patients with the polar forms of leprosy were analyzed. Macrophage activity and apoptosis were evaluated by immunohistochemistry using lysozyme, CD68, iNOS and caspase 3 as markers The nonparametric Mann-Whitney test and Spearman`s linear correlation test were used for statistical analysis The results suggest that the apoptosis rate is under the direct influence of macrophage activity in lesions of patients with the tuberculoid form In contrast, in lepromatous lesions other factors seem to induce programmed cell death, possibly TGF-beta. Further studies are necessary to identify additional factors involved in the immunopathogenesis of leprosy. (C) 2010 Elsevier Ltd. All rights reserved