Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A novel biological activity for galectin-1: Inhibition of leukocyte-endothelial cell interactions in experimental inflammation

Galectin-1 (Gal-1), the prototype of a family of β -galactoside-binding proteins, has been shown to attenuate experimental acute and chronic inflammation. In view of the fact that endothelial cells (ECs), but not human polymorphonuclear leukocytes (PMNs), expressed Gal-1 we tested here the hypothesis that the protein could modulate leukocyte-EC interaction in inflammatory settings. In vitro, human recombinant (hr) Gal-1 inhibited PMN chemotaxis and trans-endothelial migration. These actions were specific as they were absent if Gal-1 was boiled or blocked by neutralizing antiserum. In vivo, hrGal-1 (optimum effect at 0.3 μg equivalent to 20 pmol) inhibited interleukin-1β-induced PMN recruitment into the mouse peritoneal cavity. Intravital microscopy analysis showed that leukocyte flux, but not their rolling velocity, was decreased by an anti-inflammatory dose of hrGal-1. Binding of biotinylated Gal-1 to resting and post-adherent human PMNs occurred at concentrations inhibitory in the chemotaxis and transmigration assays. In addition, the pattern of Gal-1 binding was differentially modulated by PMN or EC activation. In conclusion, these data suggest the existence of a previously unrecognized function of Gal-1, that is inhibition of leukocyte rolling and extravasation in experimental inflammation. It is possible that endogenous Gal-1 may be part of a novel anti-inflammatory loop in which the endothelium is the source of the protein and the migrating PMNs the target for its anti-inflammatory action.

On Hepatitis C Virus Evolution: The Interaction between Virus and Host towards Treatment Outcome

Background:Hepatitis C is a disease spread throughout the world. Hepatitis C virus (HCV), the etiological agent of this disease, is a single-stranded positive RNA virus. Its genome encodes a single precursor protein that yields ten proteins after processing. NS5A, one of the non-structural viral proteins, is most associated with interferon-based therapy response, the approved treatment for hepatitis C in Brazil. HCV has a high mutation rate and therefore high variability, which may be important for evading the immune system and response to therapy. The aim of this study was to analyze the evolution of NS5A quasispecies before, during, and after treatment in patients infected with HCV genotype 3a who presented different therapy responses.Methods:Viral RNA was extracted, cDNA was synthesized, the NS5A region was amplified and cloned, and 15 clones from each time-point were sequenced. The sequences were analyzed for evolutionary history, genetic diversity and selection.Results:This analysis shows that the viral population that persists after treatment for most non-responder patients is present in before-treatment samples, suggesting it is adapted to evade treatment. In contrast, the population found in before treatment samples from most end-of-treatment responder patients either are selected out or appears in low frequency after relapse, therefore changing the population structure. The exceptions illustrate the uniqueness of the evolutionary process, and therefore the treatment resistance process, in each patient.Conclusion:Although evolutionary behavior throughout treatment showed that each patient presented different population dynamics unrelated to therapy outcome, it seems that the viral population from non-responders that resists the treatment already had strains that could evade therapy before it started. © 2013 Bittar et al.

Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-α/β heterodimer. Importin-α contains the NLS binding site, whereas importin-β mediates the translocation through the nuclear pore. We characterized the interactions involving importin-α during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-α is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-β (stoichiometry, 1:1; K D = 1.1 × 10 -8 M) increases the affinity for NLSs; the importin-α/β complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K D = 3.5 × 10 -8 M and 4.8 × 10 -8 M, respectively) comparable with those of a truncated importin-α lacking the autoinhibitory domain (T-antigen NLS, K D = 1.7 × 10 -8 M; nucleoplasmin NLS, K D = 1.4 × 10 -8 M). The autoinhibitory domain (as a separate peptide) binds the truncated importin-α, and the crystal structure of the complex resembles the structure of full-length importin-α. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-α and provide a quantitative description of the binding and regulatory steps during nuclear import.

Morphology and histoenzymology of eosinophilic granulocytes in the circulating blood of the turtle (Chrysemys dorbignih)

The studies on the characterization of eosinophils and neutrophils/ heterophils of turtles are contradictory. Some authors have pointed out the existence of two distinct cell types: eosinophils and heterophils. Other authors have proposed that eosinophils and heterophils may be the same cells in different stages of maturation. These interpretations are based only on a morphological analysis. In the blood of the turtle (Chrysemys dorbignih), a South American freshwater species, there are two types of granulocytes with eosinophilic staining pattern: the first with round cytoplasmic granules and the second with ellipsoidal cytoplasmic granules. In the present study by using histoenzymological methods for the analyses of enzymological cellular content, we found that the cells with round cytoplasmic granules were positive for nonspecific esterase and the cells with ellipsoidal granules were positives for acid phosphatase, alkaline phosphatase, nonspecific esterase and peroxidase. The results show that these cells are distinct cells and that the cells with ellipsoidal cytoplasmic granules have the same histoenzymological characteristics as the neutrophils/heterophils of mammalians and other vertebrates.

A complexa fisiopatologia dos episódios vasooclusivos na anemia falciforme

Hemolytic anemia and vasoocclusion are the cardinal clinical features of sickle cell anemia. Vasoocclusion is a complex process involving not only the polymerization of deoxygenated sickle hemoglobin tetramers, but also interactions between sickle erythrocytes, vascular endothelium, platelets, leukocytes, and plasma proteins. The increased adherence of sickle erythrocytes to endothelium has been implicated as an early step in vasoocclusion. Other researchers have focused on leukocytes and platelets which might also contribute to disturbed blood flow. Microvascular occlusion results in acute painful crises, whereas macrovascular occlusion seems to be the cause of organ failure. The anemia results from the markedly shortened circulatory survival of sickle erythrocytes, together with a limited erythropoietic response. The erythropoiesis increases intensively, but it is not enough to balance the increased rate of erythrocytes destruction to maintain normal levels of total erythrocytes and hemoglobin concentrations; mainly by the low oxygen affinity of hemoglobin S and increased 2,3-Diphosphoglycerate. It is very difficult to separate processes leading to anemia or to vasoocclusion. Understanding the involvement of multiple blood componentes in vasoocclusion may elucidate the clinical manifestations and complications of sickle cell anemia, and may give new insights into the preventive and curative therapy.

Inactivation of vesicular stomatitis virus through inhibition of membrane fusion by chemical modification of the viral glycoprotein

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells triggered by conformational changes in viral glycoproteins. We have demonstrated previously that modification of vesicular stomatitis virus (VSV) with diethylpyrocarbonate (DEPC) abolished conformational changes on VSV glycoprotein and the fusion reaction catalyzed by the virus. In the present study, we evaluated whether treatment with DEPC was able to inactivate the virus. Infectivity and viral replication were abolished by viral treatment with 0.5 mM DEPC. Mortality profile and inflammatory response in the central nervous system indicated that G protein modification with DEPC eliminates the ability of the virus to cause disease. In addition, DEPC treatment did not alter the conformational integrity of surface proteins of inactivated VSV as demonstrated by transmission electron microscopy and competitive ELISA. Taken together, our results suggest a potential use of histidine (His) modification to the development of a new process of viral inactivation based on fusion inhibition. © 2006 Elsevier B.V. All rights reserved.

Reproductive technology in domestic carnivorous

Background: The delay in development of artificial reproduction techniques on carnivorous could be due to countless reasons, but the lack of commercial interest is probably the most important one. The majority of canines are small structures, canidae are extremely fertile and a great number of species are adapted to domestication or captivity. Finally, the canine gamete physiology presents a difficult adaptation of technology knowledge obtained from other species. Furthermore, domestic felines are animals of company and there is no interest in reproducing them in a large scale, as it has been observed in other domestic animals, however, besides of being a valuable model for the development of in vitro techniques, the domestic cat is also used as an embryo receptor for different species of small wild felines due to physiological similarities among them, in vitro embrionary development, Review: It was reviewed the main insights about the reproductive physiology in female dogs, in vitro oocytary maturation (IVM), pregnancy and conception rate with dogs' frozen/unfrozen semen and PIV in domestic cats. The majority of mammal oocytes restart meiosis spontaneously after ovulation and reaches MII in artificial environment; in an in vitro maturation system in bovines, around 90% of oocytes complete their maturation, although its development capacity can be reduced subsequently. The success of IVM in canidae have been limited, with maturation rate varying from 0 to 58%, usually around 20%. The greatest difficulties include oocyte quality, hormonal environment, protein supplementation, cumulus / oocyte cell interaction, donor breed and age, culture systems, oxygen tension, amino acids, growth factor and sequential means. The freezing process reduces the quality of the semen, firstly because it reduces the number of living sperms and secondly because freezing produces cell modifications that could alter the sperm motility, longevity, integrity of membranes and its fertilizing capacity. Conclusion: Nowadays, several researches are being performed with the aim of increasing viability after dogs' and cats' semen is unfrozen, using extenders, cryoprotectors, freezing and unfreezing curves, addition of antioxidant substances. The aim of this text is to inform about the improvements obtained on the artificial reproduction techniques, emphasizing the oocytary maturation in female dogs, semen cryopreservation and artificial insemination in domestic dogs and cats.

Fetal-Maternal Interactions in the Synepitheliochorial Placenta Using the eGFP Cloned Cattle Model

Background: To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. Methodology/Principal Findings: Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. Conclusions/Significance: Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse. © 2013 Pereira et al.

Cytotoxic, genotoxic/antigenotoxic and mutagenic/antimutagenic effects of the venom of the wasp Polybia paulista

Hymenoptera venoms are constituted by a complex mixture of chemically or pharmacologically bioactive agents, such as phospholipases, hyaluronidases and mastoparans. Venoms can also contain substances that are able to inhibit and/or diminish the genotoxic or mutagenic action of other compounds that are capable of promoting damages in the genetic material. Thus, the present study aimed to assess the effect of the venom of Polybia paulista, a neotropical wasp, by assays with HepG2 cells maintained in culture. The cytotoxic potential of the wasp venom, assessed by the methyl thiazolyl tetrazolium assay (MTT assay), was tested for the concentrations of 10μg/mL, 5μg/mL and 1μg/mL. As these concentrations were not cytotoxic, they were used to evaluate the genotoxic (comet assay) and mutagenic potential (micronucleus test) of the venom. In this study, it was verified that these concentrations induced damages in the DNA of the exposed cells, and it was necessary to test lower concentrations until it was found those that were not considered genotoxic and mutagenic. The concentrations of 1ng/mL, 100pg/mL and 10pg/mL, which did not induce genotoxicity and mutagenicity, were used in four different treatments (post-treatment, pre-treatment, simultaneous treatment with and without incubation), in order to evaluate if these concentrations were able to inhibit or decrease the genotoxic and mutagenic action of methyl methanesulfonate (MMS). None of the concentrations was able to inhibit and/or decrease the MMS activity. The genotoxic and mutagenic activity of the venom of P. paulista could be caused by the action of phospholipase, mastoparan and hyaluronidase, which are able to disrupt the cell membrane and thereby interact with the genetic material of the cells or even facilitate the entrance of other compounds of the venom that can act on the DNA. Another possible explanation for the genotoxicity and mutagenicity of the venom can be the presence of substances able to trigger inflammatory process and, consequently, generate oxygen reactive species that can interact with the DNA of the exposed cells. © 2013 Elsevier Ltd.

Estudo dose-resposta e perfil de expressão gênica do herbicida diuron [3-(3,4-diclofenil)-1,1-dimetiluréia] em bexiga urinária de ratos wistar machos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expressão do gene Glipican 3 (Gpc3) no urotélio da bexiga de ratos expostos ao diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) por 7 dias e 20 semanas

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted urea herbicide widely used in crops of sugar cane, cotton and soybeans. In 1997, this agent has been classified by the United States Environmental Protection Agency as known/likely human carcinogen because it induced tumors in the urinary bladder and renal pelvis of rats, and breast and skin of mice exposed to 2500 ppm for feed for two years. A previous study from our group demonstrated dose-response relationship in the gene expression profile associated with severe necrosis on bladder urothelium and increased incidence of simple hyperplasia in male Wistar rats treated with different concentrations of diuron for 20 weeks. To check how early the molecular changes occurs, rats were fed for 7 days with diets containing diuron at 0, 125, 500 or 2500 ppm. The main observations recorded were urothelium ultrastructural alterations and disruptions of molecular pathways associated with cell-cell interaction and the tissue organization maintenance. Particularly, the gene Glypican 3 (Gpc3), a surface proteoglycan related to cellular adhesion and apoptosis induction, was down regulated on urothelium exposed to 2500ppm diuron for 7 days and 20 weeks. The aim of this study was validate by quantitative RT-PCR real time, the reduced Gpc3 gene expression in epithelial cells of the urinary bladder of male Wistar rats treated with different concentrations of diuron for 7 days and 20 weeks. The endogenous control of the quantitative PCR real time technique was the β-actin gene and the target was the gene Gpc3. The relative quantification (RQ) was obtained by the method of relative quantification 2-ΔΔCt . Animals exposed to diuron for 7 days or for 20 weeks presented reduction of Gpc3 gene expression compared to the control group. This reduction was statistically significant only for the 7 days study. Moreover, by comparing animals exposed for 7 days with the exposed for 20 weeks, it was ...

Eficácia e segurança da ceratoplastia endotelial no tratamento da ceratopatia bolhosa pseudofácia e afácia: revisão sistemática e metanálise

Pós-graduação em Bases Gerais da Cirurgia - FMB

Novas alternativas terapêuticas para o tratamento da Criptococose: análogos de Resveratrol e microRNAs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.