Genetic analysis of complement C1s deficiency associated with systemic lupus erythernatosus highlights alternative splicing of normal C1s gene

Deficiencies of complement proteins of the classical pathway are strongly associated with the development of autoimmune diseases. Deficiency of Clr has been observed to occur concomitantly with deficiency in Cls and 9 out of 15 reported cases presented systemic lupus erythernatosus (SLE). Here, we describe a family in which all four children are deficient in Cls but only two of them developed SLE. Hemolytic activity mediated by the alternative and the lectin pathways were normal, but classical pathway activation was absent in all children`s sera. Cls was undetectable, while in the parents` sera it was lower than in the normal controls. The levels of Clr observed in the siblings and parents sera were lower than in the control, while the concentrations of other complement proteins (C3, C4, MBL and MASP-2) were normal in all family members. Impairment of Cls synthesis was observed in the patients` fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the Cls cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of Cls mRNA transcripts in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3` splice site within intron I which increases the size of exon 2 by 87 nucleotides. (c) 2007 Elsevier Ltd. All rights reserved.

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.

A new approach to the granulation of beta-cyclodextrin inclusion complexes

Cyclodextrins (CDs) are annular oligosaccharides containing 6-12 glucose unities joined together by alpha-1,4 bonds. They have a conical-truncated shape with a lipophilic cavity in which different molecules can be included resulting in a stable inclusion complex. The cyclodextrins have been widely applied in pharmaceutical technology with the objective of increasing the solubility, stability and bioavailability of drugs in different pharmaceutical dosage forms, such as tablets. In order to obtain beta-CD tablets, liquid dispersions of drug/beta-CD are usually submitted to different drying processes, like spray-drying, freeze-drying or slow evaporation, being this dry material added to a number of excipients. However, such drying processes can generate particulate materials showing problems of flow and compressibility, needing their conversion into granulates by means of wetting with granulation liquid followed by additional drying. In this work, the main objective was to evaluate the preparation of tablets without the need of this additional drying step. For this purpose an aqueous dispersion containing acetaminophen/beta-CD complex and cornstarch was dried using a spouted bed and the obtained granules were compressed in tablets. Acetaminophen was used as model drug due to its low water solubility and the inexpensive and widely available cornstarch was chosen as excipient. Acetaminophen powder was added into a beta-cyclodextrin solution prepared in distilled water at 70 degrees C. Stirring was kept until this dispersion cooled to room temperature. Then cornstarch was added and the resulting dispersion was dried in spouted bed equipment. This material was compressed into tablets using an Erweka Korsh EKO tablet machine. This innovative approach allowed the tablets preparation process to be carried out with fewer steps and represents a technological reliable strategy to produce beta-cyclodextrin inclusion complexes tablets. (C) 2010 Elsevier By. All rights reserved.

Prostaglandin E(2)-loaded microspheres as strategy to inhibit phagocytosis and modulate inflammatory mediators release

PGE(2), an arachidonic acid metabolite produced by various type of cells regulates a broad range of physiological activities in the endocrine, cardiovascular, gastrointestinal, and immune systems, and is involved in maintaining the local homeostasis. In the immune system, PGE(2) is mainly produced by APCs and it can suppress the Th1-mediated immune responses. The aim of this study was to develop PGE(2)-loaded biodegradable MS that prolong and sustain the in vivo release of this mediator. An o/w emulsion solvent extraction-evaporation method was chosen to prepare the MS. We determined their diameters, evaluated the in vitro release of PGE(2), using enzyme immunoassay and MS uptake by peritoneal macrophages. To assess the preservation of biological activities of this mediator, we determined the effect of PGE(2) released from MS on LPS-induced TNF-alpha release by murine peritoneal macrophages. We also analyzed the effect of encapsulated PGE(2) on inflammatory mediators release from HUVECs. Finally, we studied the effect of PGE(2) released from biodegradable MS in sepsis animal model. The use of this formulation can provide an alternative strategy for treating infections, by modulating or inhibiting inflammatory responses, especially when they constitute an exacerbated profile. (C) 2008 Elsevier B.V. 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.

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.

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.

Trypanosoma cruzi: Effects of social stress in Calomys callosus a natural reservoir of infection

Social environment can represent a major source of stress affecting cortisol and/or corticosterone levels, thereby altering the immune response. We have investigated the effects of social isolation on the development of Trypanosoma cruzi infection in female Calomys callosus, a natural reservoir of this protozoan parasite. Animals were divided in groups of five animals each. The animals of one group were kept together in a single cage. In a second group, four females were kept together in a cage with one male. In the final group, five individuals were kept isolated in private cages. The isolated animals showed body weight reduction, decreased numbers of peritoneal macrophages, lower global leucocytes counts, smaller lytic antibody percentage and a significantly higher level of blood parasites compared to the other animals. Their behavior was also altered. They were more aggressive than grouped females, or females exposed to the presence of a male. These results suggest that isolation creates a distinct social behavior in which immunity is impaired and pathogenesis is enhanced. (C) 2008 Elsevier Inc. All rights reserved.

Trypanosoma cruzi: The effects of zinc supplementation during experimental infection

it is well recognized that zinc is an essential trace element, influencing growth and affecting the development and integrity of the immune system. The use of oligoelements as zinc can be considered a tool in modulating the effectiveness of the immune response. In this work zinc was daily and orally supplied in male Wistar rats infected with the Y strain of Trypanosoma cruzi. Parasiternia was evaluated and a significant reduction on blood parasites was observed. In order to check some immunological parameters peritoneal macrophages were counted revealing higher percentages for zinc supplied group. Consequently enhanced concentrations of IFN-gamma was found and for the first time NO was evaluated in T cruzi infected animals under the influence of zinc therapy, revealing enhanced concentrations when compared to. unsupplied counterparts. We conclude that zinc is able to up-regulate the host`s immune response against parasite replication. (c) 2007 Elsevier Inc. All rights reserved.

Prolactin: Does it exert an up-modulation of the immune response in Trypanosoma cruzi-infected rats?

During the course of infection by Trypanosma cruzi, the host immune system is involved in distinct, complex interactions with the endocrine system, and prolactin (PRL) is one of several hormones involved in immunoregulation. Although intensive studies attempting to understand the mechanisms that underlie Chagas` disease have been undertaken, there are still some pieces missing from this complex puzzle. Because data are scarce concerning the role of PRL involvement in Chagas` disease and taking into account the existence of crosstalk between neuroendocrine hormones and the immune system, the current study evaluates a possible up-regulation of the cellular immune response triggered by PRL in T. cruzi-infected rats and the role of PRL in reversing immunosuppression caused by the parasitic infection. The data shown herein demonstrate that PRL induces the proliferation of T lymphocytes, coupled with an activation of macrophages and the production of nitric oxide (NO), leading to a reduction in the number of blood trypomastigotes during the peak of parasitemia. During the acute phase of T. cruzi infection, an enhancement of both CD3+CD4+ and CD3+CD8+ T cell populations were observed in infected groups, with the highest numbers of these T cell subsets found in the infected group treated with PRL Because NO is a signaling molecule involved in a number of cellular interactions with components of the immune system and the neuroendocrine system, PRL can be considered an alternative hormone able to up-regulate the host`s immune system, consequently lowering the pathological effects of a T. cruzi infection. (C) 2011 Elsevier B.V. All rights reserved.

Influence of Er:YAG Laser Frequency on Dentin Caries Removal Capacity

The purposes of this study were to evaluate in vitro the influence of different frequencies of Er:YAG laser on the human dentin caries removal capacity. Thirty fragments obtained from third molars were randomly assigned into three groups (n = 10) according to the laser frequency used: 4, 6, and 10 Hz. The caries lesion (+/-1 mm deep) was induced before the irradiation by S. mutans cultures for 6 weeks. The specimens of all groups were irradiated with 200 mJ of energy in noncontact and focused mode under constant refrigeration (water flow: 2.5 mL/min). Quantitative analysis of the caries removal was performed by DIAGNOdent (TM) and the Axion Vision (TM) software. Qualitative analysis was performed by Scanning electron microscope (SEM) and light microscope (LM). Data were analyzed by ANOVA and Fishers` tests. The DIAGNOdent (TM) revealed that the caries removal was similar with 4 and 6 Hz and was superior with 10 Hz (P < 0.05). The analysis with Axion Vision (TM) software revealed that the caries removal was similar with 6 and 10 Hz and the 4 Hz group promoted the lowest caries removal. Through SEM morphologic analysis, some specimens irradiated with 4 Hz presented, under the demineralized dentin, a disorganized collagenous matrix. The LM images revealed that all frequencies used promoted irregular caries removal, being observed over preparations with 6 and 10 Hz. It can be concluded that the increase of Er:YAG laser frequency provided a higher dentin caries removal without selectivity to the disorganized dentin. Microsc. Res. Tech. 74:281-286, 2011. (C) 2010 Wiley-Liss, Inc.

Effects of dehydroepiandrosterone-sulfate (DHEA-S) and benznidazole treatments during acute infection of two different Trypanosoma cruzi strains

A significant role for hormones in regulating the balance of Th1- and Th2-associated cytokines with a role in modulating diseases has been accumulating. Previously, we reported that dehydroepiandrosterone (DHEA), the most abundant steroid hormone synthesized by the adrenal cortex, markedly reduced the blood and tissue parasites in experimentally Trypanosoma cruzi-infected rats. Based on these findings, the main purpose of this study was to investigate the effect of dehydroepiandrosterone-sulfate ester (DHEA-S) therapy alone or in combination with benznidazole (BNZ) (recommended in Brazil for the treatment of T. cruzi infection) will be effective during the acute phase of two different lineages of T. cruzi strains: type I (Y strain) and type II (Bolivia strain) of T. cruzi. Administration of either DHEA-S or BNZ alone or in combination significantly reduced the Y strain parasite load as compared with untreated. Furthermore treatment with DHEA-S resulted in Bolivia strain clearance. This protective effect of DHEA-S was associated with the host`s immune response, as evidence by enhanced levels of interferon-gamma and interleukin-2. DHEA-S treatment also increased peritoneal macrophages levels and nitrite production. DHEA-S treatment was effective in reducing the mortality rate as compared to BNZ alone or to combiner DHEA-S+BNZ treatment of T. cruzi Bolivia strain infected animals. These findings suggest that hormonal therapy may have a protective effect in the treatment of T. cruzi infection. (C) 2009 Elsevier GmbH. All rights reserved.

Immunomodulatory effects of zinc and DHEA on the Th-1 immune response in rats infected with Trypanosoma cruzi

Chagas` disease is considered the sixth most important neglected tropical disease worldwide. Considerable knowledge has been accumulated concerning the role of zinc on cellular immunity. The steroid hormone dehydroepiandrosterone (DHEA) is also known to modulate the immune system. The aims of this paper were to investigate a possible synchronization of their effects on cytokines and NO production and the resistance to Trypanosoma cruzi during the acute phase of infection. It was found that zinc, DHEA or zinc and DHEA supplementation enhanced the immune response, as evidenced by a significant reduction in parasitemia levels. Zinc and DHEA supplementation exerted additive effects on the immune response by elevation of macrophage counts, and by increasing concentrations of IFN-gamma and NO. (C) 2009 Elsevier GmbH. All rights reserved.

Enhanced protection by melatonin and meloxicam combination in experimental infection by Trypanosoma cruzi

P>The aim of this study was to evaluate a possible synergism between melatonin and meloxicam in up-regulating the immune response in male Wistar rats infected with Trypanosoma cruzi during immunosuppression phenomenon, which characterizes the acute phase of the Chagas` disease. Male Wistar rats were infected with the Y strain of T. cruzi. Experiments were performed on 7, 14 and 21 days post-infection. Several immunological parameters were evaluated including gamma-interferon (IFN-gamma), interleukin-2 (IL-2), nitric oxide (NO) and prostaglandin E(2) (PGE(2)). The combined treatment with melatonin and meloxicam significantly enhanced the release of IL-2 and INF-gamma into animals` serum, when compared with the infected control groups during the course of infection. Furthermore, the blockade of PGE(2) synthesis and the increased release of NO by macrophage cells from T. cruzi-infected animals contributed to regulate the production of Th1 subset cytokines significantly reducing the parasitaemia in animals treated with the combination of both substances. Therefore, our results suggest that the association of melatonin and meloxicam was more effective in protecting animals against the harmful actions of T. cruzi infection as compared with the treatments of meloxicam or melatonin alone.

Melatonin and dehydroepiandrosterone combination: does this treatment exert a synergistic effect during experimental Trypanosoma cruzi infection?

Previous studies showed that melatonin or dehydroepiandrosterone (DHEA) enhances the immune response against parasitic pathogens. The present study investigated the in vitro activity of melatonin combined with DHEA in a period of 24 hr during the course of in vivo T. cruzi infection. The in vitro activity of melatonin or DHEA alone, as well as together, were tested for the trypomastigote forms (doses ranging from 0.5 to 128 mu m). In vitro, neither melatonin nor DHEA alone had any activity against trypomastigote forms, although when the highest concentration of combined melatonin and DHEA was used, it was active against the trypomastigote forms of the parasite. However, for this concentration, a quite toxicity on peritoneal macrophages was observed. For in vivo evaluation, male Wistar rats were infected with the Y strain of T. cruzi. They were orally treated with 10 mg/kg body weight/day of melatonin and subcutaneously with 40 mg/kg body weight/day of DHEA. Treatment with melatonin, DHEA and the association showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection as compared to untreated animals (P < 0.05). A significant increase in the number of macrophages and nitric oxide (NO) concentrations were observed during the peak of parasitaemia with melatonin alone or combined with DHEA. However, with DHEA alone the highest concentration of NO was observed (P < 0.05). Moreover, DHEA treatment increased TNF-alpha levels during the infection (P < 0.05). These results show that melatonin, DHEA or the combination of both reduces parasitemia during the acute phase of infection. The combined action of both molecules did not exert a synergic action on the host`s ability to fight infection, and it seems that among all treatments DHEA induces a more efficient immune response.