Nitric oxide donor trans-[RuCl([15]aneN(4))NO]2+as a possible therapeutic approach for Chagas` disease

Background and purpose: Benznidazole (Bz) is the therapy currently available for clinical treatment of Chagas` disease. However, many strains of Trypanosoma cruzi parasites are naturally resistant. Nitric oxide (NO) produced by activated macrophages is crucial to the intracellular killing of parasites. Here, we investigate the in vitro and in vivo activities against T. cruzi, of the NO donor, trans-[RuCl([15]aneN(4))NO]2+. Experimental approach: Trans-[RuCl([15]aneN(4))NO]2+ was incubated with a partially drug-resistant T. cruzi Y strain and the anti-proliferative (epimastigote form) and trypanocidal activities (trypomastigote and amastigote) evaluated. Mice were treated during the acute phase of Chagas` disease. The anti-T. cruzi activity was evaluated by parasitaemia, survival rate, cardiac parasitism, myocarditis and the curative rate. Key results: Trans-[RuCl([15]aneN(4))NO]2+ was 10- and 100-fold more active than Bz against amastigotes and trypomastigotes respectively. Further, trans-[RuCl([15]aneN(4))NO]2+ (0.1 mM) induced 100% of trypanocidal activity (trypomastigotes forms) in vitro. Trans-[RuCl([15]aneN(4))NO]2+ induced permanent suppression of parasitaemia and 100% survival in a murine model of acute Chagas` disease. When the drugs were given alone, parasitological cures were confirmed in only 30 and 40% of the animals treated with the NO donor (3.33 mu mol center dot kg-1 center dot day-1) and Bz (385 mu mol center dot kg-1 center dot day-1), respectively, but when given together, 80% of the animals were parasitologically cured. The cured animals showed an absence of myocarditis and a normalisation of cytokine production in the sera. In addition, no in vitro toxicity was observed at the tested doses. Conclusions and implications: These findings indicate that trans-[RuCl([15]aneN(4))NO]2+ is a promising lead compound for the treatment of human Chagas` disease. This article is commented on by Machado et al., pp. 258-259 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00662.x and to view a related paper in this issue by Silva et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00524.x.

Exploring weak interactions to assemble a nitrosyl-ruthenium compound able to release NO under visible light irradiation

This work reports oil a novel nitrosyl-ruthenium complex hearing the azanaphthalene ligand quinazoline (qui) ill its coordination sphere. The product crystallizes with ail additional quinazoline molecule, yielding the compound cis-[Ru(bpy)(2)(qui)NO](PF(6))(3).(qui). This feature leads to all absorption band at lambda(max) = 430 nm in CH(3)CN and lambda(max) = 420 nm in phosphate buffer, which promotes the photorelease of nitric oxide under visible light irradiation (lambda > 400 nm), in 1 ethanol: 1 water (v/v) mixture or under physiological pH. Both the intensity and energy of this transition are dependent on solvent and solution pH, suggesting that the transition has a charge transfer nature, and that the association of the second quinazoline molecule with the complex is driven by weak interactions, possibly of the pi-stacking type. (C) 2009 Elsevier Ltd. All rights reserved.

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.

3`3-Ditrifluoromethyldiphenyl diselenide: A new organoselenium compound with interesting antigenotoxic and antimutagenic activities

The trace element selenium (Se), once known only for its potential toxicity, is now a well-established essential micronutrient for mammals. The organoselenium compound diphenyl diselenide (DPDS) has shown interesting antioxidant and neuroprotective activities. On the other hand, this compound has also presented pro-oxidant and mutagenic effects. The compound 3`3-ditrifluoromethyldiphenyl diselenide (DFDD), a structural analog of diphenyl diselenide, has proven antipsychotic activity in mice. Nevertheless, as opposed to DPDS, little is known on the biological and toxicological properties of DFDD. In the present study, we report the genotoxic effects of the organoselenium compound DFDD on Salmonella typhimurium, Saccharomyces cerevisiae and Chinese hamster lung fibroblasts (V79 cells). DFDD protective effects against hydrogen peroxide (H(2)O(2))-induced DNA damage in vitro are demonstrated. DFDD did not cause mutagenic effects on S. typhimurium or S. cerevisiae strains; however, it induced DNA damage in V79 cells at doses higher than 25 mu M, as detected by comet assay. DFDD protected S. typhimurium and S. cerevisiae against H(2)O(2)-induced mutagenicity, and, at doses lower than 12.5 mu M, prevented H(2)O(2)-induced genotoxicity in V79 cells. The in vitro assays demonstrated that DFDD mimics catalase activity better than DPDS, but neither presents Superoxide dismutase action. The products of the reactions of DFDD or DPDS with H(2)O(2) were different. as determined by electrospray mass spectrometry analysis (ESI-MS). These results suggest that DFDD is not mutagenic for bacteria or yeast; however, it may induce weak genotoxic effects on mammalian cells. In addition, DFDD has a protective effect against H(2)O(2)-induced damage probably by mimicking catalase activity, and the distinct products of the reaction DFDD with H(2)O(2) probably have a fundamental role in the protective effects of DFDD. (C) 2009 Elsevier B.V. All rights reserved.

Preparation and characterization of D, L-PLA loaded 17-beta-Estradiol valerate by emulsion/evaporation methods

PLA microparticles containing 17-beta-estradiol valerate were prepared by an emulsion/evaporation method in order to sustain drug release. This system was characterized concerning particle size, particle morphology and the influence of formulation and processing parameters on drug encapsulation and in vitro drug release. The biodegradation of the microparticles was observed by tissue histological analysis. Scanning electron microscopy and particle size analysis showed that the microparticles were spherical, presenting non-aggregated homogeneous surface and had diameters in the range of 718-880 nm (inert microparticles) and 3-4 mu m (drug loaded microparticles). The encapsulation efficiency was similar to 80%. Hormone released from microparticles was sustained. An in vivo degradation experiment confirmed that microparticles are biodegradable. The preparation method was shown to be suitable, since the morphological characteristics and efficiency yield were satisfactory. Thus, the method of developed microparticles seems to be a promising system for sustained release of 17-beta-estradiol.

The oleoresin secretory system in seedlings and adult plants of copaiba (Copaifera langsdorffii Desf., Leguminosae-Caesalpinioideae)

The ecological and economic importance of oleoresin produced by Copaifera langsdorffii is well established. This study aims to investigate the ontogeny, anatomy and ultrastructure of the internal glands of C. langsdorffii during plant development. Samples were processed for light and electron microscopy and a specific technique was applied to impregnate endomembranes. Internal secretory glands were observed in the hypocotyl, epicotyl and eophylls of seedlings, and in the primary stem, pulvinus, petiole, rachis and leaf blade of adult plants. Canals and cavities show differential distribution. They arise from ground meristem cells, and the lumen is first formed by schizogenesis followed by later schizolysigenous development. The dense cytoplasm of epithelial cells shows mitochondria, plastids without thylakoids, polyribosomes and endoplasmic reticulum. A periplastidial reticulum was also observed. Secretion is released by eccrine, granulocrine and holocrine processes. Lipophilic and hydrophilic compounds were histochemically detected in both canals and cavities, whereas resin was detected only in canals. The presence of these substances has been associated with plants` defences against dehydration, as well as against attacks from herbivores and pathogens, from seedling stage onwards. (C) 2011 Elsevier GmbH. All rights reserved.

Dexamethasone Effects in the Strongyloides venezuelensis Infection in A Murine Model

The aim of this study was to investigate the immunomodulatory effects of glucocorticoids on the immune response to Strongyloides venezuelensis in mice. Balb/c mice were infected with S. venezuelensis and treated with Dexamethasone (Dexa) or vehicle. Dexa treatment increased circulating blood neutrophil numbers and inhibited eosinophil and mononuclear cell accumulation in the blood, bronchoalveolar, and peritoneal fluid compared with control animals. Moreover, Dexa decreased tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-3 (IL-3), IL-4, IL-5, IL-10, and IL-12 production in the lungs and circulating immunoglobulin G1. (IgG1), IgG2a, and IgE antibody levels while increasing the overall parasite burden in the feces and intestine. Dexa treatment enhanced the fertility of female nematodes relative to untreated and infected mice. In summary, the alterations in the immune response induced by Dexa resulted in a blunted, aberrant immune response associated with increased parasite burden. This phenomenon is similar to that observed in S. stercoralis-infected humans who are taking immunosuppressive or antiinflammatory drugs, including corticosteroids.

Strongyloides venezuelensis: The antigenic identity of eight strains for the immunodiagnosis of human strongyloidiasis

The antigens of eight strains of Strongyloides venezuelensis were identified by means of the indirect immunofluorescence antibody (IFAT), enzyme-linked immunosorbent assay (ELISA) and immunoblot (IB) tests. Infective larvae (L3) from these strains were obtained from Rattus norvegicus feces cultures. For IFAT, sections of U were used while the ELISA and IB, tests were conducted with alkaline extract. Ninety serum samples were tested: 30 from patients with S. stercoralis, 30 from patients with other parasitic diseases, and 30 from healthy subjects (free of parasites). Average sensitivity and specificity among all eight strains, both for IFAT and ELISA, were, respectively, 93% and 100%. In the IB, anti-S. stercoralis IgG recognized a single antigenic fraction with 45 kDa. Serum samples from patients with S. stercoralis revealed antigens from different strains of S. venezuelensis, indicating antigenic identity for possible use in the synthesis of recombinant antigen that could be useful in immunodiagnosis and vaccine against this parasite. (C) 2008 Elsevier Inc. All rights reserved.

Phagocytosis and Nitric Oxide Levels in Rheumatic Inflammatory States in Elderly Women

Background Very few studies have investigated, in the elderly, the effect of rheumatic inflammatory states on phagocyte function and free radical production. The objective of this article is to evaluate phagocytosis by neutrophils and the production of nitric oxide (.NO) by monocytes in elderly women recruited among patients of the Brazilian Public Health System. Methods: Forty patients aged more than 60 years with rheumatic inflammatory diseases were studied. Phagocytosis was measured by flow cytometry. .NO production was measured by the total nitrite assay and conventional inflammation markers were determined. Data were analyzed with the Mann Whitney nonparametric test and P<0.05 was considered significant. Results. C-reactive protein levels and white blood cell counts were significantly higher in inflammation than in the control group (P<0.05). The phagocytosis fluorescence intensity per neutrophil and the percentual of neutrophils expressing phagocytosis were significantly higher (P<0.05) in the test than in the control group. Furthermore, there was significant .NO overproduction by monocytes, (P<0.05). Conclusion: Phagocytosis and .NO production are affected by rheumatic states. This suggests that the increased .NO levels may play a part in the increased oxidative stress in rheumatic diseases in elderly women. J. Clin. Lab. Anal. 25:47-51, 2011. (C) 2011 Wiley-Liss, Inc.

Infectivity of Strongyloides venezuelensis is influenced by variations in temperature and time of culture

The present research investigated the influence of temperature and time of larvae culture on the infectivity of Strongyloides venezuelensis. Mice were infected s.c. with 1500 larvae of S. venezuelensis maintained at 28 degrees C for three days of culture (dc), 28 degrees C for seven dc or 18 degrees C for seven dc. On days 1,3, 5, 7, 14 and 21 post-infection the animals were sacrificed and cell numbers in the blood, peritoneal cavity fluid (PCF), broncoalveolar fluid (BALF), cytokines, immunoglobulins, number of parasites and eggs/g of feces were quantified. Results demonstrated an increase in eosinophils and mononuclear cells in the blood, PCF and HALF of infected mice. Larvae at 28 degrees C/3dc induced earlier eosinophils in the PCF and HALF as opposed to larvae at 28 degrees C/7dc and 18 degrees C/7dc. Larvae at 28 degrees C/7dc induced higher synthesis of IL-4. IL-5 and IL-10 on days Sand 7 post-infection. Larvae at 28 degrees C/3dc in culture induced higher synthesis of IL-12 than larvae of seven dc, but time in culture induced better synthesis of IFN-gamma, after larval migration had ceased and only adult worms were present. Larvae at 28 degrees C/3dc in culture induced higher synthesis of IgG and IgG1 and expelled less female parasites than larvae cultivated for seven days. In conclusion, it was observed that the infectivity of S. venezuelensis is influenced by variations in temperature and time of culture. (C) 2010 Elsevier Inc. All rights reserved.

Cyclooxygenase-derived mediators regulate the immunological control of Strongyloides venezuelensis infection

The aim of this study was to define the immunoregulatory role of prostaglandins in a mouse model of Strongyloides venezuelensis infection. Strongyloides venezuelensis induced an increase of eosinophils and mononuclear cells in the blood, peritoneal cavity fluid, and bronchoalveolar lavage fluid. Treatment with the dual cyclooxygenase (COX-1/-2) inhibitors indomethacin and ibuprofen, and the COX-2-selective inhibitor celecoxib partially blocked these cellular responses and was associated with enhanced numbers of infective larvae in the lung and adult worms in the duodenum. However, the drugs did not interfere with worm fertility. Cyclooxygenase inhibitors also inhibited the production of the T-helper type 2 (Th2) mediators IL-5, IgG1, and IgE, while indomethacin alone also inhibited IL-4, IL-10, and IgG2a. Cyclooxygenase inhibitors tended to enhance the Th1 mediators IL-12 and IFN-gamma. This shift away from Th2 immunity in cyclooxygenase inhibitor-treated mice correlated with reduced prostaglandin E(2) (PGE(2)) production in infected duodenal tissue. As PGE(2) is a well-characterized driver of Th2 immunity, we speculate that reduced production of this lipid might be involved in the shift toward a Th1 phenotype, favoring parasitism by S. venezuelensis. These findings provide new evidence that cyclooxygenase-derived lipids play a role in regulating host defenses against Strongyloides, and support the exploration of eicosanoid signaling for identifying novel preventive and therapeutic modalities against these infections.

Phagocytosis, Oxidative Burst, and Produced Reactive Species are Affected by Iron Deficiency Anemia and Anemia of Chronic Diseases in Elderly

Iron and oxidative stress have a regulatory interplay. During the oxidative burst, phagocytic cells produce free radicals such as hypochlorous acid (HOCl). Nevertheless, scarce studies evaluated the effect of either iron deficiency anemia (IDA) or anemia of chronic disease (ACD) on phagocyte function in the elderly. The aim of the present study was to determine the oxidative burst, phagocytosis, and nitric oxide ((aEuro cent)NO) and HOCl, reactive species produced by monocytes and neutrophils in elderly with ACD or IDA. Soluble transferrin receptor, serum ferritin, and soluble transferrin receptor/log ferritin (TfR-F) index determined the iron status. The study was constituted of 39 patients aged over 60 (28 women and 11 men) recruited from the Brazilian Public Health System. Oxidative burst fluorescence intensity per neutrophil in IDA group and HOCl generation in both ACD and IDA groups were found to be lower (p < 0.05). The percentages of neutrophils and monocytes expressing phagocytosis in ACD group were found to be higher (p < 0.05). There was an overproduction of (aEuro cent)NO from monocytes, whereas the fundamental generation of HOCl appeared to be lower. Phagocytosis, oxidative burst, and (aEuro cent)NO and HOCl production are involved in iron metabolism regulation in elderly patients with ACD and IDA.

Counterregulation of Th2 immunity by interleukin 12 reduces host defenses against Strongyloides venezuelensis infection

The aim of this study was to investigate the role of interleukin 12 (IL-12) during Strongyloides venezuelensis infection. IL-12(-/-) and wildtype C57BL/6 mice were subcutaneously infected with 1500 larvae of S. venezuelensis. On days 7, 14, and 21 post-infection, we determined eosinophil and mononuclear cell numbers in the blood and broncoalveolar lavage fluid (BALF), Th2 cytokine secretion in the lung parenchyma, and serum antibody levels. The numbers of eggs in the feces and worm parasites in the duodena were also quantified. The eosinophil and mononuclear cell counts and the concentrations of IL-3, IL-5, IL-10, IL-13, and IgG1 and IgE antibodies increased significantly in infected IL-12(-/-) and wild-type mice as compared with uninfected controls. However, the number of eosinophils and mononuclear cells in the blood and BALF and the Th2 cytokine levels in the lungs of infected IL-12-/- mice were greater than in infected wild-type C57BL/6 mice. In addition, serum IgE and IgG1 levels were also significantly enhanced in the infected mice lacking IL-12. Meanwhile, parasite burden and fecal egg counts were significantly decreased in infected IL-12-/- mice. Together, our results showed that the absence of IL-12 upregulates the Th2 immune response, which is important for control of S. venezuelensis infection. (C) 2009 Elsevier Masson SAS. All rights reserved.

Microsatellite allele sequencing in population analyses of the South American cactophilic species Drosophila antonietae (Diptera: Drosophilidae)

Drosophila antonietae belongs to the Drosophila buzzatii cluster, a cactophilic group of species naturally endemic to South America. Morphological and genetic analyses indicate that its populations are the most homogenous in the cluster and that the diversity observed is mainly a result of variation within populations. Seven polymorphic microsatellite loci were described for this species and used in the present study to investigate the genetic diversity of natural populations of D. antonietae by both length and sequence variation. The study aimed to understand how homoplasy and null alleles affect inferences about the population history of this species and to obtain an accurate interpretation of population inferences where these loci could be applied. The results provide useful information on the interpretation of genetic data derived from the microsatellite loci described for D. antonietae and on evolutionary aspects of cactophilic Drosophila. Importantly, the results indicate that size homoplasy and null alleles do not represent significant problems for the population genetics analyses because the large amount of variability at microsatellite loci compensate the low frequency of these problems in the populations. (C) 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 573-584.

Allozymatic divergence between border populations of two cryptic species of the Drosophila buzzatii cluster species (Diptera: Drosophilidae)

Drosophila antonietae and Drosophila gouveai are allopatric, cactophilic, cryptic and endemic of South America species, which aedeagus morphology is considered the main diagnostic character. In this work, single close populations from the edge distributions of each species, located in an ""introgressive corridor"", were analyzed regarding temporal isozenzymatic genetic variability. Isocitrate dehydrogenase (Idh) appeared as a diagnostic locus between D. antonieate and D. gouveai because each population was fixed for different alleles. Moreover, several polymorphic loci showed accentuated divergence in the allele frequency, as evidenced by Nei`s l(0.3188) and D (1.1432), and also by Reynolds` genetic distance and identity (1.3207 and 0.7331, respectively). Our results showed that, in spite of the very similar external morphology, related evolutionary histories, close distributions, and events of introgression in the studied area, these cryptic species have high allozymatic differentiation, and this is discussed here. (C) 2010 Elsevier Ltd. All rights reserved.