O sistema imune dos insetos e o controle de algumas doenças parasitárias humanas

Parasitic diseases in humans, transmitted by insects, affect about 500 million people living mainly in countries of low economic power. The control of these diseases is difficult to carry out, mianly due to social and political problems, enhanced by the capacity of these organisms to develop resistance to insecticides used to for their destruction. Some recent advances in the area of insect immunology have open the possibility for abetter epidemiological control of these diseases. The immune system of these insects, as well as that of other organisms, have the ability to recognize the infecting parasites and liberate a series of reactions which stop the infection. These reactions involve the circulating cells (hemocytes) against the parasite. These cells have the ability of phagocytize and liberate the production of various humoral factors, neutralizing the infection. Some promising results, obtained by the study of the immune system of malaria-transmitting insects, the sleeping disease, and dengue, are an example of this new sanitary strategy.

Insect/Bt-cotton interactions: Are immunological variables and hemocyte ultrastructure in Podisus nigrispinus (Hemiptera: Pentatomidae) affected?

Recent studies have shown that ingestion by the army worm Spodoptera frugiperda of Cry1Ac toxin from Bt cotton promotes histochemical and ultrastructural changes in the digestive cells of the predatory pentatomid bug Podisus nigrispinus. Therefore, mindful of the changes in the midgut of the predator, which represents the first line of defence in this insect, our aim was to test the hypothesis that the consumption of Bt cotton-fed S. frugiperda by P. nigrispinus might lead to alterations in components of the immune system of P. nigrispinus. The Cry1Ac toxin level in the leaves of Bt cotton, nitric oxide, phenoloxidase activity, and total proteins were quantified by ELISA. Total and differential hemocyte counts were evaluated, and hemocyte ultrastructure analysis was undertaken. We found that ingestion of the prey fed daily with approximately 23 ± 0.70 ng g-1 Cry1Ac by wet weight of leaves, and expressed by the Bt cotton, induces small ultrastructural changes in the predator's granulocytes and plasmatocytes. However, these changes did not affect the total number and differential and humoral variables analyzed for the bug's hemocytes. © 2013 Copyright Taylor and Francis Group, LLC.

The ovarian cycle histochemistry and its relationship with hepatopancreas weight in the blue crab Callinectes danae (Crustacea: Portunidae)

Zara, F.J., Gaeta, H.H., Costa, T.M., Toyama, M.H. and Caetano, F.H. 2011. The ovarian cycle histochemistry and its relationship with hepatopancreas weight in the blue crab Callinectes danae (Crustacea: Portunidae). -Acta Zoologica (Stockholm) 00:1-13. Several studies use macroscopic patterns of the ovarian development in crustaceans. Here, we examined the relationship between ovary histochemistry, changes in gonadosomatic and hepatosomatic indices against the macroscopic pattern of the ovarian development in Callinectes danae. Animals were collected in the south coast of São Paulo State, Brazil. Ovaries were macroscopically classified as juvenile, rudimentary, developing, intermediary, mature, and rudimentary ovigerous. Samples were fixed in 4% paraformaldehyde, processed for historesin, and stained with HE, protein, and neutral and acid polysaccharides detection. The juvenile oocytes are not enclosed by follicular cells and have fewer yolk nuclei being less intense in PAS reactivity than rudimentary oocytes. Developing oocytes show yolk granules and thick follicular cells. Yolk granules were positive for proteins and neutral polysaccharides. The intermediary stage is marked by a qualitative increase in yolk granules and the onset of chorion formation. In mature oocytes, cytoplasm is completely filled by yolk granules and the chorion is completely formed. Ovigerous ovaries have several atretic follicles and large quantities of hemocytes in the process of tissue reorganization. In C. danae, the changes in cell, goandosomatic and hepatosomatic indices coinciding with macroscopic observations and any combination of different macroscopic stages in a single pattern should be avoided. © 2011 The Royal Swedish Academy of Sciences.

Caracterização morfofisiológica dos hemócitos do Diplópodo Rhinocricus padbergi antes e após exposição a substrato contendo lodo de esgoto

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação da toxicidade de resíduos industriais e urbanos aplicados na agricultura

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Impacto geno e citotóxico em populações do caranguejo-uçá, Ucides cordatus (Linnaeus, 1763) (Crustacea, Brachyura, Ucididae), em manguezais do estado de São Paulo, Brasil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metodologia da criação de Galleria mellonella para uso como modelo de infecção e efeitos de Lactobacillus rhamnosus inativado pelo calor in vivo e in vitro, desafiados por Staphylococcus aureus e Escherichia coli

Pós-graduação em Biopatologia Bucal - ICT

Detecção molecular de Ehrlichia canis em cães e em órgãos de seus respectivos carrapatos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detecção molecular de Ehrlichia canis em cães e em órgãos de seus respectivos carrapatos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Spatial competition induces the mobilization of morula cells in the colonial ascidian Didemnum perlucidum (Tunicata: Didemnidae)

The effects of spatial competition among colonial marine organisms are often evident in the contact zones between colonies. These effects are especially pronounced when the interaction results in overgrowth or necrosis of one of the competitors. Ascidians, one of the dominant taxonomic groups in subtidal sessile communities, have specialized morula cells that provide a defense against microbial infections. Injuries resulting from interspecific competitive interactions might also act as a stimulus for this defensive mechanism. Therefore, we expected to see the recruitment of morula cells in tissues near competitor contact zones. To test the hypothesis that spatial competition elicits this immune response, we placed colonies of the ascidian Didemnum perlucidum from southeastern Brazil in four different types of competitive situations: (1) overgrowth of the competitor, (2) stand-off interactions, (3) overgrowth by the competitor, and (4) free of competitors. Our results indicate that competitive interactions increase the population of morula cells in contact zones, as more cells were observed in interactions that resulted in the overgrowth of individuals of D. perlucidum, and fewer cells were observed in colonies that were free of competitors. We identified the defensive function of the morula cells by showing the presence of the enzyme phenoloxidase within its vacuoles. Phenoloxidase is a widespread enzyme among animals and plants, and is frequently used in defense by synthesizing toxic quinones from polyphenol substrates. This is the first study to document the presence of morula cells in didemnid ascidians and the mobilization of these cells by spatial competition by heterospecifics, and one of the first studies to identify phenoloxidase activity in morula cells.

Synthesis and properties of cyclic gomesin and analogues

Gomesin (Gm) was the first antimicrobial peptide (AMP) isolated from the hemocytes of a spider, the Brazilian mygalomorph Acanthoscurria gomesiana. We have been studying the properties of this interesting AMP, which also displays anticancer, antimalarial, anticryptococcal and anti-Leishmania activities. In the present study, the total syntheses of backbone-cyclized analogues of Gm (two disulfide bonds), [Cys(Acm)2,15]-Gm (one disulfide bond) and [Thr2,6,11,15,d-Pro9]-Gm (no disulfide bonds) were accomplished, and the impact of cyclization on their properties was examined. The consequence of simultaneous deletion of pGlu1 and Arg16-Glu-Arg18-NH2 on Gm antimicrobial activity and structure was also analyzed. The results obtained showed that the synthetic route that includes peptide backbone cyclization on resin was advantageous and that a combination of 20% DMSO/NMP, EDC/HOBt, 60?degrees C and conventional heating appears to be particularly suitable for backbone cyclization of bioactive peptides. The biological properties of the Gm analogues clearly revealed that the N-terminal amino acid pGlu1 and the amidated C-terminal tripeptide Arg16-Glu-Arg18-NH2 play a major role in the interaction of Gm with the target membranes. Moreover, backbone cyclization practically did not affect the stability of the peptides in human serum; it also did not affect or enhanced hemolytic activity, but induced selectivity and, in some cases, discrete enhancements of antimicrobial activity and salt tolerance. Because of its high therapeutic index, easy synthesis and lower cost, the [Thr2,6,11,15,d-Pro9]-Gm analogue remains the best active Gm-derived AMP developed so far; nevertheless, its elevated instability in human serum may limit its therapeutic potential. Copyright (c) 2012 European Peptide Society and John Wiley & Sons, Ltd.

EROD activity and genotoxicity in the seabob shrimp Xiphopenaeus kroyeri exposed to benzo[a]pyrene (BaP) concentrations

Seabob shrimp Xiphopenaeus kroyeri is a marine species that lives in shallow waters of coastal environments, often impacted by polycyclic aromatic hydrocarbons (PAH) pollution. In the present study, seabob shrimp were exposed for 96 h to benzo[a]pyrene (BaP) at the nominal concentrations of 100, 200, 400 and 800 microg.L-1. Animals of the control groups were exposed either to clean water or to the BaP-carrier (DMSO). At the end of the exposures, muscle tissues were sampled for BaP uptake assessment and hepatopancreas and hemolymph for EROD enzyme activity and hemocytes DNA damage, respectively. EROD activity and DNA damage increased significantly as a function of BaP exposure concentrations. Significant correlations between BaP uptake and both EROD activity and DNA damage suggest that they can be used as suitable tools for integrated levels of study on the biomarkers of PAH exposure.

The spider acylpolyamine Mygalin is a potent modulator of innate immune responses

Mygalin is an antibacterial molecule isolated froth the hemocytes of the spider Acanthoscurria gomesiana. It was identified as bis-acylpolyamine spermidine. We evaluated the modulator effects of synthetic Mygalin in the innate immune response. We demonstrate that Mygalin induces IFN-gamma synthesis by splenocytes increasing the nitrite secretion by splenocytes and macrophages. A specific inhibitor of iNOS abrogated Mygalin-induced nitrite production in macrophages independent of IFN-gamma activation. In addition, Mygalin-activated macrophages produced TNF-alpha but not IL-1 beta, demonstrating that Mygalin does not act directly on the inflammasome. Furthermore, this compound did not affect spontaneous or Concanavalin A-induced proliferative responses by murine splenocytes and did not induce IL-5 or apoptosis of splenocytes or bone marrow-derived macrophages. These data provide evidence that Mygalin modulates the innate immune response by inducing IFN-gamma and NO synthesis. The combined immune regulatory and antibacterial qualities of Mygalin should be explored as a strategy to enhance immune responses in infection. (C) 2012 Elsevier Inc. All rights reserved.

Innate Immunity in Insects, Function and Regulation of Hemolin from Hyalophora cecropia

Insects are useful models for the study of innate immune reactions and development. The distinction between recognition mechanisms preceding the breakdown of apoptotic cells during metamorphosis, and the breakdown of cells in response to infections, is unclear. Hemolin, a Lepidopteran member of the immunoglobulin superfamily, is a candidate molecule in self/nonself recognition. This thesis investigates hemolin function and hemolin gene regulation at a molecular level. We investigated the binding and cell adhesion properties of hemolin from H. cecropia and demonstrated that the proteins could homodimerize in presence of calcium. Moreover, a higher molecular weight membrane form of hemolin was present on hemocytes. These results, taken together with an earlier finding that soluble hemolin inhibits hemocyte adhesion, indicated that the secreted hemolin could modulate hemocyte aggregation in a competitive manner in the blood. In addition, hemolin was expressed in different tissues and at different developmental stages. Since hemolin is expressed both during development and during the immune response, its different regulatory factors must act in concert. We found that the third intron contains an enhancer, through which Dif, C/EBP and HMGI synergistically activate a reporter construct in vitro. We concluded that the enhancer is used during infection, since the κB-site is crucial for an immune response. Interestingly, we also found that the active form of the steroid hormone, ecdysone, induces the hemolin gene transcription in vivo, and in addition, acts synergistically during bacterial infection. Preliminary in vivo results indicate a secondary effect of ecdysone and the importance of hormone receptor elements in the upstream promoter region of hemolin. To explore the use of Drosophila as a genetic tool for understanding hemolin function and regulation, we sought to isolate the functional homologue in this species. A fly cDNA library in yeast was screened using H. cecropia hemolin as bait. The screen was not successful. However, it did lead to the discovery of a Drosophila protein with true binding specificity for hemolin. Subsequent characterization revealed a new, highly conserved gene, which we named yippee. Yippee is distantly related to zinc finger proteins and represents a novel family of proteins present in numerous eukaryotes, including fungi, plants and humans. Notably, when the Drosophila genome sequence was revealed, no hemolin orthologue could be detected. Finally, an extensive Drosophila genome chip analysis was initiated. The goal was to investigate the Drosophila immune response, and, in contrast to earlier studies of artificially injected flies, to examine a set of natural microbes, orally and externally applied. In parallel experiments viruses, bacteria, fungi and parasites were compared to unchallenged controls. We obtained a unique set of genes that were up-regulated in the response to the parasite Octosporea muscadomesticae and to the fungus Beauveria bassiana. We expect both down-regulated and up-regulated genes to serve as a source for the discovery of new effector molecules, in particular those that are active against parasites and fungi.

A SMO inhibitor drug reduces the progenitor cell's maintenance in the Drosophila hematopoietic organ: a novel model to study Chronic Myelogenous Leukemia relapse

The chronic myeloid leukemia complexity and the difficulties of disease eradication have recently led to the development of drugs which, together with the inhibitors of TK, could eliminate leukemia stem cells preventing the occurrence of relapses in patients undergoing transplantation. The Hedgehog (Hh) signaling pathway positively regulates the self-renewal and the maintenance of leukemic stem cells and not, and this function is evolutionarily conserved. Using Drosophila as a model, we studied the efficacy of the SMO inhibitor drug that inhibit the human protein Smoothened (SMO). SMO is a crucial component in the signal transduction of Hh and its blockade in mammals leads to a reduction in the disease induction. Here we show that administration of the SMO inhibitor to animals has a specific effect directed against the Drosophila ortholog protein, causing loss of quiescence and hematopoietic precursors mobilization. The SMO inhibitor induces in L3 larvae the appearance of melanotic nodules generated as response by Drosophila immune system to the increase of its hemocytes. The same phenotype is induced even by the dsRNA:SMO specific expression in hematopoietic precursors of the lymph gland. The drug action is also confirmed at cellular level. The study of molecular markers has allowed us to demonstrate that SMO inhibitor leads to a reduction of the quiescent precursors and to an increase of the differentiated cells. Moreover administering the inhibitor to heterozygous for a null allele of Smo, we observe a significant increase in the phenotype penetrance compared to administration to wild type animals. This helps to confirm the specific effect of the drug itself. These data taken together indicate that the study of inhibitors of Smo in Drosophila can represent a useful way to dissect their action mechanism at the molecular-genetic level in order to collect information applicable to the studies of the disease in humans.