Defense reactions of Dermatobia hominis (Diptera : Cuterebridae) larval hemocytes

The defense reactions against biological (Histoplasma capsulatum and Escherichia coli) and non-biological materials (China ink and nylon thread) were tested in vivo in third instar larvae of Dermatobia hominis. The cellular defense performed by larval hemocytes was observed under electron microscopy. China ink particles were phagocytosed by granular cells 5 h after injection. E. coli cells were internalized by granular cells as early as 5 min after injection and totally cleared 180 min post-injection, when many hemocytes appeared disintegrated and others in process of recovering. H. capsulatum yeasts provoked, 24 h after being injected, the beginning of nodule formation. Nylon thread was encapsulated 24 h after the introduction into the hemocoel. Our results suggest that granular cells were the phagocytic cells and also the responsible for the triggering of nodule and capsule formation. In the presence of yeasts cells and nylon thread, they released their granules that chemotactically attracted the plasmatocytes that on their turn, flattened to surround and isolate the foreign material.

Oral Immune Defense against Chronic Hyperplastic Candidosis

Candida yeast species are widespread opportunistic microbes, which are usually innocent opportunists unless the systemic or local defense system of the host becomes compromised. When they adhere on a fertile substrate such as moist and warm, protein-rich human mucosal membrane or biomaterial surface, they become activated and start to grow pseudo and real hyphae. Their growth is intricately guided by their ability to detect surface defects (providing secure hiding , thigmotropism) and nutrients (source of energy, chemotropism). The hypothesis of this work was that body mobilizes both non-specific and specific host defense against invading candidal cells and that these interactions involve resident epithelial cells, rapidly responding non-specific protector neutrophils and mast cells as well as the antigen presenting and responding den-dritic cell lymphocyte plasma cell system. It is supposed that Candida albicans, as a result of dar-winistic pressure, has developed or is utilizing strategies to evade these host defense reactions by e.g. adhering to biomaterial surfaces and biofilms. The aim of the study was to assess the host defense by taking such key molecules of the anti-candidal defense into focus, which are also more or less characteristic for the main cellular players in candida-host cell interactions. As a model for candidal-host interaction, sections of chronic hyperplastic candidosis were used and compared with sections of non-infected leukoplakia and healthy tissue. In this thesis work, neutrophil-derived anti-candidal α-defensin was found in the epithelium, not only diffusely all over in the epithelium, but as a strong α-defensin-rich superficial front probably able to slow down or prevent penetration of candida into the epithelium. Neutrophil represents the main host defence cell in the epithelium, to which it can rapidly transmigrate from the circulation and where it forms organized multicellular units known as microabscesses (study I). Neutrophil chemotactic inter-leukin-8 (IL-8) and its receptor (IL-8R) were studied and were surprisingly also found in the candidal cells, probably helping the candida to keep away from IL-8- and neutrophil-rich danger zones (study IV). Both leukocytes and resident epithelial cells contained TLR2, TLR4 and TLR6 receptors able to recognize candidal structures via utilization of receptors similar to the Toll of the banana fly. It seems that candida can avoid host defence via stimulation of the candida permissive TLR2 instead of the can-dida injurious TLR4 (study V). TLR also provides the danger signal to the immune system without which it will not be activated to specifically respond against candidal antigens. Indeed, diseased sites contained receptor activator of nuclear factor kappa B ligand (RANKL; II study), which is important for the antigen capturing, processing and presenting dendritic cells and for the T lymphocyte activation (study III). Chronic hyperplastic candidosis provides a disease model that is very useful to study local and sys-temic host factors, which under normal circumstances restrain C. albicans to a harmless commensal state, but failure of which in e.g. HIV infection, cancer and aging may lead to chronic infection.

Comparison of the Ability of Partially N-Acetylated Chitosans and Chitooligosaccharides to Elicit Resistance Reactions in Wheat Leaves1

Chitin, a linear polysaccharide composed of (1→4)-linked 2-acetamido-2-deoxy-β-d-glucopyranose (GlcNAc) residues, and chitosan, the fully or partially N-acetylated, water-soluble derivative of chitin composed of (1→4)-linked GlcNAc and 2-amino-2-deoxy-β-d-glucopyranose (GlcN), have been proposed as elicitors of defense reactions in higher plants. We tested and compared the ability of purified (1→4)-linked oligomers of GlcNAc (tetramer to decamer) and of GlcN (pentamer and heptamer) and partially N-acetylated chitosans with degrees of acetylation (DA) of 1%, 15%, 35%, 49%, and 60% and average degrees of polymerization between 540 and 1100 to elicit phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition, and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, nonwounded wheat (Triticum aestivum L.) leaves. Purified oligomers of (1→4)-linked GlcN were not active as elicitors, whereas purified oligomers of (1→4)-linked GlcNAc with a degree of polymerization ≥ 7 strongly elicited POD activities but not PAL activities. Partially N-acetylated, polymeric chitosans elicited both PAL and POD activities, and maximum elicitation was observed with chitosans of intermediate DAs. All chitosans but not the chitin oligomers induced the deposition of lignin, the appearance of necrotic cells exhibiting yellow autofluorescence under ultraviolet light, and macroscopically visible necroses; those with intermediate DAs were most active. These results suggest that different mechanisms are involved in the elicitation of POD activities by GlcNAc oligomers, and of PAL and POD activities by partially N-acetylated chitosan polymers and that both enzymes have to be activated for lignin biosynthesis and ensuing necrosis to occur.

Ozone-induced responses in Arabidopsis thaliana: the role of salicylic acid in the accumulation of defense-related transcripts and induced resistance.

Exposure of Arabidopsis thaliana to ozone results in the expression of a number of defense-related genes that are also induced during a hypersensitive response. A potential common link between the activation of defense gene expression during a hypersensitive response and by ozone treatment is the production of active oxygen species and the accumulation of hydrogen peroxide. Here we report that salicylic acid accumulation, which can be induced by hydrogen peroxide and is required for the expression of both a hypersensitive response and systemic acquired resistance, is also required for the induction of some, but not all, ozone-induced mRNAs examined. In addition, we show that ozone exposure triggers induced resistance of A. thaliana to infection with virulent phytopathogenic Pseudomonas syringae strains. Infection of transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of salicylic acid, or npr1 mutant plants, which are defective in the expression of systemic acquired resistance at a step downstream of salicylic acid, demonstrated that the signaling pathway activated during ozone-induced resistance overlaps with the systemic acquired resistance activation pathway and is salicylic acid dependent. Interestingly, plants expressing salicylate hydroxylase exhibited increased sensitivity to ozone exposure. These results demonstrate that ozone activates at least two distinct signaling pathways, including a salicylic acid dependent pathway previously shown to be associated with the activation of pathogen defense reactions, and that this latter pathway also induces a protective response to ozone.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca²⁺ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components. © 2008 Elsevier Inc. All rights reserved.

Role of glutamate NMDA receptors and nitric oxide located within the periaqueductal gray on defensive behaviors in mice confronted by predator

The midbrain periaqueductal gray (PAG) is part of the brain system involved in active defense reactions to threatening stimuli. Glutamate N-methyl-d-aspartate (NMDA) receptor activation within the dorsal column of the PAG (dPAG) leads to autonomic and behavioral responses characterized as the fear reaction. Nitric oxide (NO) has been proposed to be a mediator of the aversive action of glutamate, since the activation of NMDA receptors in the brain increases NO synthesis. We investigated the effects of intra-dPAG infusions of NMDA on defensive behaviors in mice pretreated with a neuronal nitric oxide synthase (nNOS) inhibitor [N omega-propyl-l-arginine (NPLA)], in the same midbrain site, during a confrontation with a predator in the rat exposure test (RET). Male Swiss mice received intra-dPAG injections of NPLA (0.1 or 0.4 nmol/0.1 mu l), and 10 min later, they were infused with NMDA (0.04 nmol/0.1 mu l) into the dPAG. After 10 min, each mouse was placed in the RET. NMDA treatment enhanced avoidance behavior from the predator and markedly increased freezing behavior. These proaversive effects of NMDA were prevented by prior injection of NPLA. Furthermore, defensive behaviors (e.g., avoidance, risk assessment, freezing) were consistently reduced by the highest dose of NPLA alone, suggesting an intrinsic effect of nitric oxide on defensive behavior in mice exposed to the RET. These findings suggest a potential role of glutamate NMDA receptors and NO in the dPAG in the regulation of defensive behaviors in mice during a confrontation with a predator in the RET.

Physical environment modulates the behavioral responses induced by chemical stimulation of dorsal periaqueductal gray in mice

In order to investigate the relationship between behaviors elicited by chemical stimulation of the dorsal periaqueductal gray (dorsal PAG) and spontaneous defensive behaviors to a predator, the excitatory amino acid D,L-homocysteic acid (5 nmol in 0.1 mu l), was infused into the dorsal PAG and behavioral responses of mice were evaluated in two different situations, a rectangular novel chamber or the Mouse Defense Test Battery (MDTB) apparatus. During a 1-min period following drug infusion, more jumps were made in the chamber than in the MDTB runway but running time and distance traveled were significantly higher in the runway. Animals were subsequently tested using the standard MDTB procedure (anti-predator avoidance, chase and defensive threat/attack). No drug effects on these measures were significant. In a further test in the MDTB apparatus, the pathway of the mouse during peak locomotion response was blocked 3 times by the predator stimulus (anesthetized rat) to determine if the mouse would avoid contact. Ninety percent of D,L-homocysteic treated animals made direct contact with the stimulus (rat), indicating that D,L-homocysteic-induced running is not guided by relevant (here, threat) stimuli. These results indicate that running as opposed to jumping is the primary response in mice injected with D,L-homocysteic into the dorsal PAG when the environment enables flight. However, the lack of responsivity to the predator during peak locomotion suggests that D,L-homocysteic-stimulation into the dorsal PAG does not induce normal antipredator flight. (c) 2006 Published by Elsevier B.V.

Role of glutamate NMDA receptors and nitric oxide located within the periaqueductal gray on defensive behaviors in mice confronted by predator

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

Effects of intra-PAG infusion of ovine CRF on defensive behaviors in Swiss-Webster mice

The midbrain dorsal periaqueductal gray (DPAG) is part of the brain defensive system involved in active defense reactions to threatening stimuli. Corticotrophin releasing factor (CRF) is a peptidergic neurotransmitter that has been strongly implicated in the control of both behavioral and endocrine responses to threat and stress. We investigated the effect of the nonspecific CRF receptor agonist, ovine CRF (oCRF), injected into the DPAG of mice, in two predator-stress situations, the mouse defense test battery (MDTB), and the rat exposure test (RET). In the MDTB, oCRF weakly modified defensive behaviors in mice confronted by the predator (rat); e.g. it increased avoidance distance when the rat was approached and escape attempts (jump escapes) in forced contact. In the RET, drug infusion enhanced duration in the chamber while reduced tunnel and surface time, and reduced contact with the screen which divides the subject and the predator. oCRF also reduced both frequency and duration of risk assessment (stretch attend posture: SAP) in the tunnel and tended to increase freezing. These findings suggest that patterns of defensiveness in response to low intensity threat (RET) are more sensitive to intra-DPAG oCRF than those triggered by high intensity threats (MDTB). Our data indicate that CRF systems may be functionally involved in unconditioned defenses to a predator, consonant with a role for DPAG CRF systems in the regulation of emotionality. (c) 2006 Elsevier B.V. All rights reserved.

Biomarker responses as indication of contaminant effects in Oreochromis niloticus

The current study investigated oxidative stress parameters (enzymes activities, metallothionein content and lipid peroxidation) in freshwater fish, Oreochromis niloticus, tilapia exposure to Monjolinho River (in 4 months of year: January, April, July and November). One critical site in Monjolinho River (site B) was assessed in comparison to a reference site (site A). Water pH and oxygen concentration was lower than that recommended by CONAMA (Brazilian National Environmental Committee), resolution 357/2005 for protection of aquatic communities, and ammonium and the metals Cu, Zn, Mn and Fe (on all months) concentrations were higher than the maximum concentration recommended. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were significantly decreased in liver and muscle in tila. pia from Monjolinho River, throughout the year, in relation to reference except in gills that SOD activity increased. Glutathione S-transferase (GST) activity was significantly increased in liver of the tilapia from Monjolinho River in all sites, in relation to reference except in gills that GST activity increased in July and decreased in November, suggesting that GST activity could be induced to neutralize the pollutants toxicity. On the other hand, GST activity was significantly decreased in white muscle indicating a toxic effect of pollutants, resulting in a decreased ability of tilapia to perform defense reactions associated to GSTs. The decrease of catalase (CAT) activity in gills of the O. niloticus together with the increase of SOD activity, could explain the increased lipid peroxidation (LPO) level in this organ. Metallothionein levels in liver and gills were significantly high in all sites. Results indicate that the exposure to metals caused severe damage to tissues; despite the consensually assumed antioxidant induction as a sign of exposure to contaminants the effects seem in part to be mediated by suppression of antioxidant system with SOD, CAT and GPx as potential candidates for tissues toxicity biomarkers of pollutants. (c) 2012 Elsevier Ltd. All rights reserved.

Molecular genetic causes of columnar growth in apple (Malus x domestica)

The columnar growth habit of apple is interesting from an economic point of view as the pillar-like trees require little space and labor. Genetic engineering could be used to speed up breeding for columnar trees with high fruit quality and disease resistance. For this purpose, this study dealt with the molecular causes of this interesting phenotype. The original bud sport mutation that led to the columnar growth habit was found to be a novel nested insertion of a Gypsy-44 LTR retrotransposon on chromosome 10 at 18.79 Mb. This subsequently causes tissue-specific differential expression of nearby downstream genes, particularly of a gene encoding a 2OG-Fe(II) oxygenase of unknown function (dmr6-like) that is strongly upregulated in developing aerial tissues of columnar trees. The tissue-specificity of the differential expression suggests involvement of cis-regulatory regions and/or tissue-specific epigenetic markers whose influence on gene expression is altered due to the retrotransposon insertion. This eventually leads to changes in genes associated with stress and defense reactions, cell wall and cell membrane metabolism as well as phytohormone biosynthesis and signaling, which act together to cause the typical phenotype characteristics of columnar trees such as short internodes and the absence of long lateral branches. In future, transformation experiments introducing Gypsy-44 into non-columnar varieties or excising Gypsy-44 from columnar varieties would provide proof for our hypotheses. However, since site-specific transformation of a nested retrotransposon is a (too) ambitious objective, silencing of the Gypsy-44 transcripts or the nearby genes would also provide helpful clues.

The GATA factor Serpent is required for the onset of the humoral immune response in Drosophila embryos

Innate immunity in Drosophila is characterized by the inducible expression of antimicrobial peptides. We have investigated the development and regulation of immune responsiveness in Drosophila embryos after infection. Immune competence, as monitored by the induction of Cecropin A1-lacZ constructs, was observed first in the embryonic yolk. This observation suggests that the yolk plays an important role in the humoral immune response of the developing embryo by synthesizing antimicrobial peptides. Around midembryogenesis, the response in the yolk was diminished. Simultaneously, Cecropin expression became inducible in a large number of cells in the epidermis, demonstrating that late-stage embryos can synthesize their own antibiotics in the epidermis. This production likely serves to provide the hatching larva with an active antimicrobial barrier and protection against systemic infections. Cecropin expression in the yolk required the presence of a GATA site in the promoter as well as the involvement of the GATA-binding transcription factor Serpent (dGATAb). In contrast, neither the GATA site nor Serpent were necessary for Cecropin expression in the epidermis. Thus, the inducible immune responses in the yolk and in the epidermis can be uncoupled and call for distinct sets of transcription factors. Our data suggest that Serpent is involved in the distinction between a systemic response in the yolk/fat body and a local immune response in epithelial cells. In addition, the present study shows that signal transduction pathways controlling innate and epithelial defense reactions can be dissected genetically in Drosophila embryos.

Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells1

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca2+ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca2+ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.

Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca2+ channels. Inhibition of the oxidative response impaired Cl− efflux, K+ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.