Enantioselective analysis of propranolol and 4-hydroxypropranolol by CE with application to biotransformation studies employing endophytic fungi

A CE method is described for the enantioselective analysis of propranolol (Prop) and 4-hydroxypropranolol (4-OH-Prop) in liquid Czapek medium with application in the study of the enantioselective biotransformation of Prop by endophytic fungi. The electrophoretic conditions previously optimized were as follows: an uncoated fused-silica capillary, 4%w/v carboxymethyl-beta-CD in 25 mmol/L triethylamine/phosphoric acid (H(3)PO(4)) buffer at pH 9 as running electrolyte and 17 kV of voltage. UV detection was carried out at 208 nm. Liquid-liquid extraction using diethyl ether: ethyl acetate (1:1 v/v) as extractor solvent was employed for sample preparation. The calibration curves were linear over the concentration range of 0.25-10.0 mu g/mL for each 4-OH-Prop enantiomer and 0.10-10.0 mu g/mL for each Prop enantiomer (r >= 0.995). Within-day and between-day relative standard deviations and relative errors for precision and accuracy were lower than 15% for all the enantiomers. Finally, the validated method was used to evaluate Prop biotransformation in its mammalian metabolite 4-OH-Prop by some selected endophytic fungi. The screening of five strains of endophytic fungi was performed and all of them could biotransform Prop to some extent. Specifically, Glomerella cingulata (VA1) biotransformed 47.8% of (-)-(S)-Prop to (-)-(S)-4-OH-Prop with no formation of (+)-(R)4-OH-Prop in 72 h of incubation.

Box-Behnken design for the optimization of an enantioselective method for the simultaneous analysis of propranolol and 4-hydroxypropranolol by CE

An experimental design optimization (Box-Behnken design, BBD) was used to develop a CE method for the simultaneous resolution of propranolol (Prop) and 4-hydroxypropranolol enantiomers and acetaminophen (internal standard). The method was optimized using an uncoated fused silica capillary, carboxymethyl-beta-cyclodextrin (CM-beta-CD) as chiral selector and triethylamine/phosphoric acid buffer in alkaline conditions. A BBD for four factors was selected to observe the effects of buffer electrolyte concentration, pH, CM-beta-CD concentration and voltage on separation responses. Each factor was studied at three levels: high, central and low, and three center points were added. The buffer electrolyte concentration ranged from 25 to 75 mM, the pH ranged from 8 to 9, the CM-beta-CD concentration ranged from 3.5 to 4.5%w/v, and the applied run voltage ranged from 14 to 20 W. The responses evaluated were resolution and migration time for the last peak. The obtained responses were processed by Minitab (R) to evaluate the significance of the effects and to find the optimum analysis conditions. The best results were obtained using 4%w/v CM-beta-CD in 25 mM triethylamine/H(3)PO(4) buffer at pH 9 as running electrolyte and 17 kV of voltage. Resolution values of 1.98 and 1.95 were obtained for Prop and 4-hydroxypropranolol enantiomers, respectively. The total analysis time was around of 15 min. The BBD showed to be an adequate design for the development of a CE method, resulting in a rapid and efficient optimization of the pH and concentration of the buffer, cyclodextrin concentration and applied voltage.

Trypanosoma cruzi: The effects of zinc supplementation in the immune response during the course of experimental disease

Zinc is an essential nutritional component required for normal development and maintenance of immune functions. The possible effects of zinc in upregulating the host immune response during the acute and chronic phases of experimental Chagas` disease were evaluated. In young, infected and Zn-supplemented animals, higher concentrations of IFN-gamma and NO were observed. During the chronic phase, decreased concentrations of NO and IFN-gamma were found for older infected animals that received Zn supplementation. For young animals, hearts from Zn-supplemented groups displayed reduced inflammatory infiltrate, heart weight and number of amastigote burdens. For older, infected and Zn-supplemented animals amastigote nests were absent with reduced inflammatory cell infiltrate. This study identifies a potentially novel therapeutic approach that could control the parasite load during acute phase of disease, consequently preventing the deleterious, parasite-elicited responses observed during chronic phase. (C) 2008 Elsevier Inc. All rights reserved.

Trypanosoma cruzi: Orchiectomy and dehydroepiandrosterone therapy in infected rats

The ability of gonadal hormones to influence and induce diverse immunological functions during the course of a number of parasitic infections has been extensively studied in the latest decades. Dehydroepiandrosterone and its sulfate are the most abundant steroid hormones secreted by the human adrenal cortex and are considered potent immune-activators. The effects of orchiectomy on the course of Trypanosoma cruzi infection in rats, treated and untreated with DHEA were examined, by comparing blood and cardiac parasitism, macrophage numbers, nitric oxide and IFN-gamma levels. Orchiectomy enhanced resistance against infection with elevated numbers of macrophages, enhanced concentrations of NO and IFN-gamma and reduced amastigote burdens in heart when compared to control animals. DHEA replacement exerted a synergistic effect, up-modulating the immune response. Male sex steroids appear to play fundamental role in determining the outcome of disease, through the regulation and modulation of the activity of the immune response. (C) 2008 Elsevier Inc. All rights reserved.

Trypanosoma cruzi: Effects of adrenalectomy during the acute phase of experimental infection

Glucocorticoid hormones have been implicated as an important modulator of Trypanosoma cruzi pathogenesis. Since adrenal steroid hormones play a fundamental role in modulating the immune response, we hypothesized that adrenalectomy affect the course of the experimental T. cruzi infection. This study was undertaken to determine the effects of adrenalectomy during the acute phase of T cruzi infection. Blood and tissue parasitism, macrophages, nitric oxide (NO) production and IFN-gamma were evaluated in male Wistar rats infected with the Y strain of T. cruzi. Our results show that adrenalectomized rats displayed increased number of blood and heart parasites accompanied by decreases in the total number of peritoneal macrophages and IFN-gamma when compared to controls. Adrenalectomy also reduced the levels of NO released from peritoneal macrophages of infected animals. These results suggest that adrenal corticosteroid insufficiency due to adrenalectomy could be considered an important factor during development of acute phases of experimental Chagas` disease, enhancing pathogenesis through disturbance of the host`s immune system. (C) 2008 Published by Elsevier Inc.

Melatonin enhances pro-inflammatory cytokine levels and protects against Chagas disease

Pro-inflammatory and modulatory cytokines have an essential role in host defense against human and murine Trypanosoma cruzi infection. Control of T. cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. Melatonin has been proposed to regulate the immune system by affecting cytokine production in immunocompetent cells, enhancing the production of several T helper (Th)1 cytokines. The aims of this work were to evaluate in rats, the influences of exogenous melatonin treatment on T. cruzi-infected host`s immune responses. With this in mind, several immunological parameters were analyzed, including tumor necrosis factor-alpha, gamma-interferon, interleukin-12, nitric oxide (NO) and macrophage count. The melatonin therapy was provided in one of two different treatment regimens, that is, either beginning 7 days prior to infection or concomitant with the infection. Both treatments triggered an up-regulation of the immune response, with the concomitant treatment being more effective; in this case all cytokines studied, with exception of NO, displayed enhanced concentrations and there was a higher number of peritoneal macrophages, which displayed reduced concentrations under melatonin therapy. We conclude that melatonin plays a pivotal role in up-regulating the Th1 immune response thus controlling parasite replication.

Effect of zinc supplementation in pregnant mice during experimental Trypanosoma cruzi infection

Zinc is an essential micronutrient and has significant effects on human growth, development, and immune function. Zinc supplementation or deficiency may affect the course of infection. Zinc enhances immune response against a wide range of viral, bacterial, and parasitic pathogens. In the present study, we investigated the effects of zinc sulphate (ZnSO(4)) supplementation (20 mg/kg/day) during pregnancy in mice, Swiss Webster strain infected by the Y strain of Trypanosoma cruzi. Oral supplementation of zinc sulphate in pregnant and non-pregnant infected animals did not affect the count of blood parasites as well as tissue parasitism in the heart, liver, and spleen. Zinc supplementation did not alter female body weight, the length of fetuses and neonates, placental size/weight and mortality rate. Among zinc supplied animals, no significant plasmatic zinc concentrations were observed. Concerning to tissue zinc concentrations, only the liver displayed enhanced values as compared to other organs. For placental parasitism, zinc supplied group displayed a significant decrease in amastigote burdens (P < 0.05). However due to the reduced number of parasite burdens in placenta of animals supplied with zinc, these data suggest that zinc was partially effective in up-regulating the host`s immune response against parasite, probably attenuating the infection in fetuses. (C) 2010 Elsevier Ltd. All rights reserved.

DHEA and testosterone therapies in Trypanosoma cruzi-infected rats are associated with thymic changes

The ability of the gonadal hormones to influence diverse immunological functions during the course of several infections has been extensively studied in the latest decades. Testosterone has a suppressive effect on immune response of vertebrates and increases susceptibility toward numerous parasitic diseases. Dehydroepiandrosterone is an abundant steroid hormone secreted by the human adrenal cortex and it is considered potent immune-activator. In this paper, it was examined the effects of DHEA and testosterone supplementation in the thymic atrophy in rats infected with Trypanosoma cruzi, by comparing blood parasitism, thymocyte proliferation, TNF-alpha and IL-12 levels. Our data point in the direction that DHEA treatment triggered enhanced thymocyte proliferation as compared to its infected counterparts and reduced production of TNF-alpha during the acute phase of infection. Oppositely, the lowest values for cells proliferation and IL-12 concentrations were reached in testosterone-supplied animals. The combined treatment testosterone and DHEA improves the effectiveness of the host`s immune response, reducing blood parasites and the immunosuppressive effects of male androgens besides increasing IL-12 concentrations and decreasing TNF-alpha levels. (C) 2010 Elsevier Ltd. 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.

Trypanosoma cruzi: Dehydroepiandrosterone (DHEA) and immune response during the chronic phase of the experimental Chagas` disease

Dehydroepiandrosterone (DHEA) has long been considered as a precursor for many steroid hormones. It also enhances the immune responses against a wide range of viral, bacterial, and parasitic pathogens. The aims of this work were to evaluate the influences of exogenous DHEA treatment on Wistar rats infected with the Y strain of Trypanosoma cruzi during the acute and its influence on the chronic phase of infection. Animals were subcutaneous treated with 40 mg/kg body weight/day of DHEA. DHEA treatment promoted increased lymphoproliferative responses as well as enhanced concentrations of NO and IL-12. So, we point in the direction that our results validate the utility of the use of DHEA as an alternative therapy candidate against T cruzi. (C) 2009 Published by Elsevier B.V.

A new thrombospondin-related anonymous protein homologue in Neospora caninum (NcMIC2-like1)

Neospora caninum is an Apicomplexan protozoan that has the dog as a definitive host and cattle (among other animals) as intermediate hosts. It causes encephalopathy in dogs and abortion in cows, with significant loss in worldwide livestock. As any Apicomplexan, the parasite invades the cells using proteins contained in the phylum-specific organelles, like the micronemes, rhoptries and dense granules. The aim of this study was the characterization of a homologue (denominated NcMIC2-like1) of N. caninum thrombospondin-related anonymous protein (NcMIC2), a micronemal protein previously shown to be involved in the attachment and connection with the intracellular motor responsible for the active process of invasion. A polyclonal antiserum raised against the recombinant NcMIC2-like1 functional core (thrombospondin and integrin domains) recognized the native form of NcMIC2-like1, inhibited the in vitro invasion process and localized NcMIC2-like1 at the apical complex of the parasite by confocal immunofluorescence, indicating its micronemal localization. The new molecule, NcMIC2-like1, has features that differentiates it from NcMIC2 in a substantial way to be considered a homologue dagger.

Worker demography in a large-colony, swarm-founding wasp

Neotropical swarm-founding wasps build nests enclosed in a covering envelope, which makes it difficult to count individual births and deaths. Thus, knowledge of worker demography is very limited for swarm-founding species compared with that for independent-founding species. In this study, we explored the worker demography of the swarm-founding wasp Polybia paulista, the colony size of which usually exceeds several thousand adults. We considered each wasp colony as an open-population and estimated the survival probability, recruitment rate, and population size of workers using the developments of the Cormack-Jolly-Seber model. We found that capture probability varied considerably among the workers, probably due to age polyethism and/or task specialization. The daily survival rate of workers was high (around 0.97) throughout the season and was not related to the phase of colony development. On the other hand, the recruitment rate ranged from 0 to 0.37, suggesting that worker production was substantially less important than worker survival in determining worker population fluctuations. When we compared survival rates among worker groups of one colony, the mean daily survival rate was lower for founding workers than for progeny workers and tended to be higher in progeny workers that emerged in winter. These differences in survivorship patterns among worker cohorts would be related to worker foraging activity and/or level of parasitism.

Temporal dynamic of the relationship between the parasitic isopod Aporobopyrus curtatus (Crustacea: Isopoda: Bopyridae) and the anomuran crab Petrolisthes armatus (Crustacea: Decapoda: Porcellanidae) in southern Brazil

The prevalence of the parasite Aporobopyrus curtatus in Petrolisthes armatus from southern Brazil was determined, and the effect the parasite had on host reproduction was evaluated. Of all 775 crabs sampled in Araca region from March 2005 to July 2006, 3.2% presented bopyrid parasites. All the parasitized individuals had one branchial chamber occupied by two mature parasites, with no preference for the right or left chamber. Male and female hosts were infested in equal proportions. Parasitized juveniles, large individuals and ovigerous females were not found in our study. The absence of parasitized ovigerous females seems to be insufficient evidence to support the hypothesis of parasitic castration and would require a histological study to confirm their reproductive death. The percentage of infestation observed in our study (3.1%) is lower than the one found in other studies and it could indicate the existence of factor(s) regulating the density of A. curtatus in the Araca region. At least in this population, the low but constant presence of the bopyrid A. curtatus population did not appear to have a negative effect on the porcellanid population, and parasitized individuals did not play a significant role in the natural history of P. armatus.

Decision rules for egg recognition are related to functional roles and chemical cues in the queenless ant Dinoponera quadriceps

The capacity to distinguish colony members from strangers is a key component in social life. In social insects, this extends to the brood and involves discrimination of queen eggs. Chemical substances communicate colony affiliation for both adults and brood; thus, in theory, all colony members should be able to recognize fellow nestmates. In this study, we investigate the ability of Dinoponera quadriceps workers to discriminate nestmate and non-nestmate eggs based on cuticular hydrocarbon composition. We analyzed whether cuticular hydrocarbons present on the eggs provide cues of discrimination. The results show that egg recognition in D. quadriceps is related to both age and the functional role of workers. Brood care workers were able to distinguish nestmate from non-nestmate eggs, while callow and forager workers were unable to do so.

A new social parasite in the ant genus Ectatomma F. Smith (Hymenoptera, Formicidae, Ectatomminae)

Ectatomma parasiticum Feitosa & Fresneau, a new ant species socially parasitic on Ectatomma tuberculatum (Olivier), is described from gynes discovered in Apazapan, state of Veracruz, Mexico, and reared in the laboratory. Ectatomma parasiticum is the first social parasite described in the Ectatomminae. This species can be distinguished from its host by morphological and behavioral features characteristic of the inquilines known in other ant subfamilies including reduced size, thickened petiole, and agonistic interactions with host species.