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BACKGROUND: Clinical and experimental analyses have identified a central role for IgE/FcεRI/mast cells in promoting IgE-mediated anaphylaxis. Recent data from human studies suggest that bacterial infections can alter susceptibility to anaphylaxis. OBJECTIVE: We examined the effect of LPS exposure on the induction of IgE-mast cell (MC) mediated reactions in mice. METHODS: C57BL/6 WT, tlr4-/- and IL10-/- mice were exposed to LPS, and serum cytokines (TNF and IL-10) were measured. Mice were subsequently treated with anti-IgE, and the symptoms of passive IgE-mediated anaphylaxis, MC activation, Ca2+ -mobilization and the expression of FcεRI on peritoneal MCs were quantitated. RESULTS: We show that LPS exposure of C57BL/6 WT mice constraints IgE-MC-mediated reactions. LPS-induced suppression of IgE-MC-mediated responses was TLR-4-dependent and associated with increased systemic IL-10 levels, decreased surface expression of FcεRI on MCs and loss of sensitivity to IgE activation. Notably, LPS-induced desensitization of MCs was short term with MC sensitivity to IgE reconstituted within 48 hours, which was associated with recapitulation of FcεRI expression on the MCs. Mechanistic analyses revealed a requirement for IL-10 in LPS-mediated decrease in MC FcεRI surface expression. CONCLUSIONS & CLINICAL RELEVANCE: Collectively, these studies suggest that LPS-induced IL-10 promotes the down-regulation of MC surface FcεRI expression and leads to desensitization of mice to IgE-mediated reactions. These studies indicate that targeting of the LPS-TLR-4-IL-10 pathway may be used as a therapeutic approach to prevent adverse IgE-mediated reactions.
Assuntos
Imunoglobulina E/imunologia , Lipopolissacarídeos/imunologia , Mastócitos/imunologia , Anafilaxia/imunologia , Anafilaxia/metabolismo , Animais , Cálcio/metabolismo , Degranulação Celular/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hematócrito , Interleucina-10/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Receptores de IgE/genética , Receptores de IgE/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismoRESUMO
Salmonella is a leading cause of bacterial foodborne illness. We report the collaborative investigative efforts of US and Canadian public health officials during the 2013-2014 international outbreak of multiple Salmonella serotype infections linked to sprouted chia seed powder. The investigation included open-ended interviews of ill persons, traceback, product testing, facility inspections, and trace forward. Ninety-four persons infected with outbreak strains from 16 states and four provinces were identified; 21% were hospitalized and none died. Fifty-four (96%) of 56 persons who consumed chia seed powder, reported 13 different brands that traced back to a single Canadian firm, distributed by four US and eight Canadian companies. Laboratory testing yielded outbreak strains from leftover and intact product. Contaminated product was recalled. Although chia seed powder is a novel outbreak vehicle, sprouted seeds are recognized as an important cause of foodborne illness; firms should follow available guidance to reduce the risk of bacterial contamination during sprouting.
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Surtos de Doenças , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/epidemiologia , Intoxicação Alimentar por Salmonella/epidemiologia , Salmonella/fisiologia , Salvia/microbiologia , Sementes/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Canadá/epidemiologia , Criança , Pré-Escolar , Feminino , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Salmonella/genética , Intoxicação Alimentar por Salmonella/microbiologia , Estados Unidos/epidemiologia , Adulto JovemRESUMO
Sex therapy, a specialized form of psychotherapy for sexual dysfunctions, combines the three main therapeutic approaches, cognitive-behavioral, family and psychodynamic in an integrated approach. The treatment emphasis is first placed on the sexual symptom and then, if necessary, on understanding the underlying intrapsychic and interpersonal aspects of the disorder. In addition to work on the body and fantasies, sex therapy has integrated in recent years a number of innovative approaches: combination therapies, internet therapy, pain therapy and mindfulness. Sexual disorders can be difficult to treat. It is, therefore, important to take into account the role of biological, psychological, relational and cultural factors.
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Terapia Cognitivo-Comportamental , Aconselhamento Sexual , Disfunções Sexuais Fisiológicas/terapia , Disfunções Sexuais Psicogênicas/terapia , HumanosRESUMO
The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin. It is unclear whether neutrophils also express the C3aR, but need a costimulus provided by eosinophils for certain biological responses, or whether neutrophils lack the C3aR and respond to C3a via a secondary stimulus generated by eosinophils, i.e., by an indirect mode. In the present study, polyclonal antiserum raised against the second extracellular loop of the C3aR was used to characterize C3aR expression on peripheral blood leukocytes. For high degree purification of neutrophils, a negative selection method was established that decreased the contamination with CD9(bright+) eosinophils down to <0.2%. Flow cytometric analyses, functional assays, and binding assays on highly purified neutrophils confirmed C3aR expression and coupling. Monocytes were identified as an additional C3aR-positive cell population of the peripheral blood. The expression of the C3aR on eosinophils could be confirmed. In contrast, the receptor could not be detected on unchallenged B or T lymphocytes (or lymphocyte-derived Raji cells).
Assuntos
Linfócitos B/química , Complemento C3a/metabolismo , Glicoproteínas de Membrana , Monócitos/química , Neutrófilos/química , Receptores de Complemento/análise , Linfócitos T/química , Animais , Antígenos CD/análise , Northern Blotting , Cálcio/metabolismo , Complemento C3a/farmacologia , Citometria de Fluxo , Humanos , RNA Mensageiro/análise , Coelhos , Ratos , Tetraspanina 29RESUMO
With the aid of monoclonal antibody (mAb) 2625, raised against the lipopolysaccharide (LPS) of Legionella pneumophila serogroup 1, subgroup OLDA, we isolated mutant 811 from the virulent wild-type strain RC1. This mutant was not reactive with mAb 2625 and exhibited an unstable phenotype, since we observed an in vitro and in vivo switch of mutant 811 to the mAb 2625-positive phenotype, thus restoring the wild-type LPS. Bactericidal assays revealed that mutant 811 was lysed by serum complement components, whereas the parental strain RC1 was almost serum resistant. Moreover, mutant 811 was not able to replicate intracellularly in macrophage-like cell line HL-60. In the guinea pig animal model, mutant 811 exhibited significantly reduced ability to replicate. Among recovered bacteria, mAb 2625-positive revertants were increased by fourfold. The relevance of LPS phase switch for pathogenesis of Legionella infection was further corroborated by the observation that 5% of the bacteria recovered from the lungs of guinea pigs infected with the wild-type strain RC1 were negative for mAb 2625 binding. These findings strongly indicate that under in vivo conditions switching between two LPS phenotypes occurs and may promote adaptation and replication of L. pneumophila. This is the first description of phase-variable expression of Legionella LPS.
Assuntos
Legionella pneumophila/patogenicidade , Lipopolissacarídeos/imunologia , Virulência/genética , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/imunologia , Divisão Celular/genética , Mapeamento de Epitopos , Feminino , Cobaias , Células HL-60 , Humanos , Memória Imunológica/imunologia , Legionella pneumophila/genética , Doença dos Legionários/microbiologia , Lipopolissacarídeos/química , Pulmão/microbiologia , Masculino , Microscopia Imunoeletrônica , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/imunologia , SorologiaRESUMO
ABSTRACT Isolates of Stemphylium vesicarium causing brown spot of pear can be distinguished from nonpathogenic isolates of S. vesicarium from pear or from other hosts on the basis of distinctive amplified fragment length polymorphism fingerprinting profiles. DNA fragments specific for isolates pathogenic to pear were identified and a quantitative polymerase chain reaction (PCR) was developed on the sequence from one of these specific DNA loci. This TaqMan PCR has a high sensitivity with a dynamic range for reliable quantification between 1 ng and 100 fg of DNA. The method detected pear-pathogenic isolates of S. vesicarium originating from four different European countries and various regions within those countries. No cross-reaction was found with either the nonpathogenic isolates of S. vesicarium tested or isolates belonging to other Stemphylium spp. or related fungi. The pathogen was detected on leaves with brown-spot symptoms originating from six different locations in The Netherlands, Italy, and Spain. Pear-pathogenic S. vesicarium populations were monitored on crop residues in two Dutch orchards between October 2007 and October 2008. Brown spot had been observed at both orchards at the end of the growing season of 2007. In one location, pear-pathogenic S. vesicarium was detected only sporadically on crop residues and no brown-spot symptoms were observed on fruit in 2008. At the other location, a pathogenic population was found on fallen pear leaves and on other crop residues but this population decreased during winter. From the beginning of the growing season in 2008 onward, the pathogen population could not be detected and the disease incidence was only 0.6%. The TaqMan PCR will allow more detailed studies on epidemiology of brown spot and on the effect of disease control measures.
Assuntos
Ascomicetos/fisiologia , Pyrus/microbiologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Ascomicetos/genética , Ascomicetos/isolamento & purificação , DNA Fúngico/genética , Reação em Cadeia da PolimeraseRESUMO
Cadaveric renal transplants suffer frequently from delayed graft function, which is associated with increased risk for long-term graft survival loss. One-third of kidney grafts that are stored in current organ preservation solutions experience delayed graft function, demonstrating the urgent need for improvement. Although ischaemic graft injury is complex in nature, complement activation is considered important to the process. Here we show that pharmacological targeting of the complement 5a receptor (C5aR) during cold ischaemia has a protective effect on early kidney graft survival, inflammation and apoptosis in a mouse model of syngeneic kidney transplantation. Graft survival of kidneys that were stored in University of Wisconsin solution in the presence of a C5aR antagonist increased from 29% to 57%. Increased graft survival was associated with less tubular damage and apoptosis, protection from sustained C5aR expression and decreased production of tumour necrosis factor-alpha and macrophage inflammatory protein-2. In a translational approach, we determined C5aR expression in paediatric living-related and cadaveric allografts. C5aR expression was significantly higher in all compartments of kidneys from cadaveric compared with kidneys from living-related donors. C5aR expression in cadaveric kidneys correlated positively with cold ischaemia time, renal dysfunction and the frequency of apoptotic tubular cells, suggesting a novel role for C5a in delayed graft function pathogenesis. Supplementing organ preservation solutions with C5aR inhibitors may improve early graft function following cadaveric kidney transplantation.
Assuntos
Sobrevivência de Enxerto , Transplante de Rim , Rim/patologia , Preservação de Órgãos/métodos , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Animais , Apoptose , Criança , Complemento C5a/metabolismo , Histocitoquímica , Humanos , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos , Soluções para Preservação de Órgãos/farmacologia , Receptor da Anafilatoxina C5a/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Coloração e Rotulagem , Transplante IsogênicoRESUMO
OBJECTIVE: Enlargement of the junctional zone (JZ) on T2-weighted resonance imaging of the uterus has recently been established as the major criterion for adenomyosis in patients with endometriosis. This study was conducted to analyse the extent of adenomyosis using magnetic resonance imaging (MRI) and relate it to the duration of dysmenorrhoea. STUDY DESIGN: This was a prospective study of 70 patients presenting with the complaint of severe dysmenorrhoea. Forty patients (57%) reported dysmenorrhoea as their major complaint and 30 patients (43%) suffered additionally from infertility. Group I (n=40) consisted of patients with dysmenorrhoea of between 1 and 10 years' duration, group II (n=30) consisted of patients with dysmenorrhoea of longer than 11 years' duration. All patients underwent laparoscopy to detect the presence and degree of endometriosis, and all patients underwent T2-weighted resonance imaging of the uterus to detect the extent of adenomyosis by measurement of the "junctional zone". RESULTS: In group I, adenomyosis could be detected via MRI in 21 patients (52.5%), while 19 patients (47.5%) showed no signs of adenomyosis. By contrast, in group II a distinct enlargement of the JZ, as the major radiological criterion of adenomyosis, could be observed in 26 patients (87%), while only 4 patients (13%) revealed no signs of adenomyosis (p=0.04). The mean thickness of the JZ was significantly enlarged in group II (11.07 mm) compared with group I (6.38 mm; p<0.0001). The prevalence of adenomyosis in endometriosis after dysmenorrhoea of more than 11 years' duration was 87%. CONCLUSIONS: In deep infiltrating endometriosis, a correlation between a specific localisation and dysmenorrhoea can often not be found. Recently, endometriosis and adenomyosis have been believed to result from a common uterine disease, the dislocation of the basal endometrium. Our data clearly show that dysmenorrhoea of long duration in patients who have had endometriosis for over a threshold value of 11 years is significantly related to adenomyosis of the uterus. Hence, evaluation of adenomyosis using MRI should become a standard procedure in cases of dysmenorrhoea and endometriosis. Severe dysmenorrhoea of long duration should always focus clinical interest on adenomyosis of the uterus.
Assuntos
Dismenorreia/patologia , Endometriose/patologia , Imageamento por Ressonância Magnética , Adulto , Progressão da Doença , Dismenorreia/diagnóstico , Dismenorreia/etiologia , Endometriose/complicações , Endometriose/diagnóstico , Endométrio/patologia , Feminino , Humanos , Estudos Prospectivos , Fatores de TempoRESUMO
ABSTRACT Naturally occurring populations of Fusarium avenaceum, F. culmorum, F. graminearum, F. poae, and Microdochium nivale were studied in two field experiments from anthesis in June 2003 until harvest in crops of winter wheat, and subsequently during 10 months after harvest until June 2004 on their residues exposed on the soil surface under field conditions. The dynamics of the different pathogens were estimated by quantifying the amount of DNA present in wheat tissues using TaqMan-polymerase chain reaction. While colonization of grain by Fusarium spp. and M. nivale was low, high amounts of DNA of F. avenaceum, F. graminearum, and F. culmorum were found in ear residues, internodes, and nodes of the mature crop. Amounts of DNA of pathogens decreased significantly during the following 10 months in residues of internodes and nodes, but not in residues of stem bases. Knowledge on population dynamics of pathogens will help to develop preventive measures aimed at reduction of inoculum sources of head blight pathogens.
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ABSTRACT A spatially explicit model describing saprophytic colonization of dead cyclamen leaf tissue by the plant-pathogenic fungus Botrytis cinerea and the saprophytic fungal antagonist Ulocladium atrum was constructed. Both fungi explore the leaf and utilize the resources it provides. Leaf tissue is represented by a two-dimensional grid of square grid cells. Fungal competition within grid cells is modeled using Lotka-Volterra equations. Spatial expansion into neighboring grid cells is assumed proportional to the mycelial density gradient between donor and receptor cell. Established fungal biomass is immobile. Radial growth rates of B. cinerea and U. atrum in dead cyclamen leaf tissue were measured to determine parameters describing the spatial dynamics of the fungi. At temperatures from 5 to 25 degrees C, B. cinerea colonies expanded twice as rapidly as U. atrum colonies. In practical biological control, the slower colonization of space by U. atrum thus needs to be compensated by a sufficiently dense and even distribution of conidia on the leaf. Simulation results confirm the importance of spatial expansion to the outcome of the competitive interaction between B. cinerea and U. atrum at leaf scale. A sensitivity analysis further emphasized the importance of a uniform high density cover of vital U. atrum conidia on target leaves.
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In recent years a plethora of data has accumulated directing toward an important role of polypeptides C3a and C5a and its degradation product C5adesArg, summarized as anaphylatoxins (ATs), in microbial host defense and immune regulation. The ATs exert their various biologic functions by interacting with specific C3a- and C5a-receptors present on cells of myeloid origin, epithelial cells, smooth muscle cells as well as on activated B- and T-cells. Activation of AT receptors mediates signal transduction pathways triggering a variety of proinflammatory events. However, by interacting with the cytokine- and chemokine network C3a and C5a exhibit also anti-inflammatory properties. In this review the focus is on the pathogenetic role of the ATs in sepsis, immune complex disease, delayed type hypersensitivity and asthma. Discussed are data from animal models in which the ATs are blocked by specific C3a or C5a inhibitors or from mice with genetic deletions of the specific receptors of either C3a or C5a/C5adesArg.
Assuntos
Anafilatoxinas/imunologia , Asma/etiologia , Hipersensibilidade Tardia/etiologia , Doenças do Complexo Imune/etiologia , Sepse/etiologia , Complemento C3a/imunologia , Complemento C5a/imunologia , Complemento C5a des-Arginina/imunologia , Humanos , Modelos ImunológicosRESUMO
The contribution of either the complement system or the activation of Fc receptors for IgG (FcyRs) to the inflammatory response in immune complex (IC) disease is puzzling. A series of studies has been performed in mice with engineered deficiencies of either FcgammaRs, the complement components C3, C4 or the C5a receptor. In addition, different C5-deficient mice strains have been evaluated. Mice with gene targeted disruption of the gamma-subunit, which mediates surface expression and signal transduction of the high affinity Fc receptor type I for IgG (FcgammaRI), the low affinity receptor Fc receptor type III for IgG (FcgammaRIII) and the high affinity receptor type I for IgE (IgepsilonRI), showed an impaired inflammatory response in the reverse passive Arthus reaction in skin, peritoneum and lung. These data suggest, that the activation of FgammaRs is the initial event triggering the inflammatory cascade in IC disease. On the other hand, C5aR deficient mice are either protected from tissue injury induced by ICs, as in the lung, or the degree of the inflammatory response is markedly attenuated, as in peritoneum and skin. A detailed analysis of data obtained with the different knock-out strains revealed that both the activation of the complement system as well as the activation of different effector cells via FcgammaRs contribute to the inflammatory sequelae leading to tissue destruction in IC disease. The relative contributions of FcgammaRI or FcgammaRIII and the main effector cells through which these receptors mediate their effector functions are tissue dependent. The activation of the C5a receptor pathway appears to be the prominent contribution of the complement system.
Assuntos
Reação de Arthus/etiologia , Reação de Arthus/imunologia , Proteínas do Sistema Complemento/metabolismo , Receptores de IgG/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas do Sistema Complemento/deficiência , Proteínas do Sistema Complemento/genética , Humanos , Doenças do Complexo Imune/etiologia , Doenças do Complexo Imune/imunologia , Inflamação/etiologia , Inflamação/imunologia , Pneumopatias/etiologia , Pneumopatias/imunologia , Camundongos , Camundongos Knockout , Peritonite/etiologia , Peritonite/imunologia , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Receptores de IgG/deficiência , Receptores de IgG/genéticaRESUMO
The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35-40 was randomly mutated and the resulting C5a[35-40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A35E36R37A38S39R40], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q35E36R37I38L39N40] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.
Assuntos
Antígenos CD/metabolismo , Complemento C5a des-Arginina/genética , Receptores de Complemento/metabolismo , Bacteriófagos , Sítios de Ligação/genética , Ligação Competitiva , Complemento C5a des-Arginina/metabolismo , Biblioteca Gênica , Humanos , Receptor da Anafilatoxina C5a , Células U937RESUMO
Xenogeneic liver transplantation in the discordant guinea pig (gp) to rat model results in hyperacute rejection within a few minutes, which is due to activation of the complement system. Currently no antibodies against gp complement factors are available, which allow activation of the gp complement system in serum or complement deposition in tissue to be detected. To close this gap, we started developing single chain Fvs (scFvs) against gpC5 and gpC5a. We generated a combinatorial library of scFv antibodies comprising the variable heavy and light chain repertoire from mice immunized with gpC5. Out of this library we selected several antibodies against gpC5 and C5a after four and six rounds of biopanning, respectively. Selected gpC5-specific scFvs were purified by metal affinity chromatography followed by size exclusion chromatography or by affinity chromatography using Protein L. Purified scFvs were able to inhibit gp complement system in a hemolytic assay and to detect gpC5 deposition in tissue. A surface plasmon resonance based assay on BIAcore was established, with which the C5 concentration in gp serum was determined to 240 microg/ml. As at least 0.04% of the normal gpC5 concentration can be detected, the test provides a powerful tool to investigate the development and the consequence of a hybrid complement system after liver xenotransplantation from gp to rat.
Assuntos
Anticorpos Heterófilos/isolamento & purificação , Complemento C5/imunologia , Complemento C5a/imunologia , Imunologia de Transplantes , Doença Aguda , Sequência de Aminoácidos , Animais , Anticorpos Heterófilos/biossíntese , Anticorpos Heterófilos/genética , Afinidade de Anticorpos , Ativação do Complemento , Complemento C5/genética , Complemento C5a/genética , Proteínas Inativadoras do Complemento/biossíntese , Proteínas Inativadoras do Complemento/genética , Proteínas Inativadoras do Complemento/isolamento & purificação , Reações Cruzadas , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/imunologia , Cobaias , Transplante de Coração/imunologia , Imunização , Transplante de Fígado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ratos , Transplante HeterólogoRESUMO
The nanoscaled ternary transition-metal fluorides Li3MF6 (M = V, Fe, Mn) and Li2NiF4 are promising candidates for cathode materials in high-voltage lithium-ion batteries. The fluorolytic route to these compounds relies on thermal decomposition of a hitherto uncharacterised precursor mixture produced from acetylacetonates and hydrofluoric acid. By addition of pyridine, different cationic, electroneutral and anionic complexes containing the motifs [MFn]((3-n)+) (n = 0-4) have been trapped and characterised by single-crystal X-ray diffraction and IR spectroscopy. Based on the results, a model of successive and incomplete fluorination is proposed for the speciation and formation of the precursor. The decomposition of the latter has been monitored via thermogravimetry (TG) and differential scanning calorimetry (DSC).
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Mycotoxins including aflatoxins, deoxynivalenol, fumonisins and ochratoxin A are among the main fungal secondary metabolites detected as natural contaminants in South America in different commodities such as peanuts (aflatoxins), cereals (deoxynivalenol and fumonisins) or grapes (ochratoxin A). Different strategies including crop rotation, tillage practices, fungicide application and planting less susceptible cultivars are used in order to reduce the impact of these mycotoxins in both food and feed chains. The development of fungicide resistance in many fungal pathogens as well as rising of public concern on the risks associated with pesticide use led to the search for alternative environmentally friendly methods. Biological control of plant pathogens and toxigenic fungi offers an alternative that can complement chemical control in the frame of an integrated pest management to reduce the impact of mycotoxins in the food and feed chains. The advances made in Argentina on reducing the impact of toxigenic fungi and mycotoxins in peanut, grapes and cereals using the biocontrol strategy are summarised. Native bacteria, yeasts and filamentous fungi have been selected to evaluate them as potential biocontrol agents. Field trials showed that Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were effective at reducing deoxynivalenol accumulation in wheat. The application of Clonostachys rosea isolates on wheat stubble reduced Fusarium colonisation on the stubble. Bacillus amyloliquefaciens and Microbacterium oleovorans showed good activity to control both Fusarium verticillioides growth and the accumulation of fumonisins at pre-harvest stage in maize. Control of toxigenic Aspergillus flavus and aflatoxin accumulation in peanuts was achieved using a native atoxigenic Aspergillus flavus strain based on competitive exclusion of the toxigenic strains. Kluyveromyces thermotolerans strains were used as biocontrol agents to reduce the impact of Aspergillus section Nigri and ochratoxin A accumulation in grapes.
Assuntos
Arachis/microbiologia , Agentes de Controle Biológico , Grão Comestível/microbiologia , Micotoxinas/análise , Vitis/microbiologia , Aflatoxinas/análise , Argentina , Aspergillus flavus/isolamento & purificação , Bacillus subtilis/fisiologia , Brevibacillus/fisiologia , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Fumonisinas/análise , Fusarium/isolamento & purificação , Ocratoxinas/análise , Zea mays/microbiologiaRESUMO
Binding and effector domains of the human anaphylatoxin C5a have been determined by either site directed mutagenesis or synthetic peptide studies. However, the lack of specific selection methods, which allow direct investigation of C5a-C5a-receptor interaction made these studies laborious. To overcome these limitations we have constructed a novel Fos-C5a expressed on the tip of a filamentous phage. To guarantee for a free C-terminus which is required for C5a activity C5a cDNA was cloned into the phagemid vector pJuFo. Helper phage infection of pJuFc-C5a transformed cells resulted in a mutant phage displaying Fos-C5a on its surface. However studies with Bt2cAMP differentiated U937 cells revealed that phage displayed Fos-C5a is functional inactive. Subsequently we replaced a nonconserved cysteine residue at position 27 by alanine and obtained Fos-C5aAla27. Both the purified and the phage displayed Fos-C5aAla27 proteins were functional active and induced enzyme release from differentiated U937 cells. In addition, purified Fos-C5aAla27 exhibited the same binding profile as compared to rhC5a. Fos-C5aAla27 displaying phages were mixed with phage harboring only the pJuFo plasmid at a ratio of 10(6). After four successive rounds of panning on differentiated U937 cells Fos-C5aAla27 phages were enriched to 100% as shown by C5a-specific ELISA. We expect this approach to prove helpful for studying C5a-C5a-receptor interactions. i.e. to screen C5a libraries for high affinity binders with agonistic or antagonistic properties directly on cells.
Assuntos
Antígenos CD/metabolismo , Bacteriófagos/genética , Complemento C5a/metabolismo , Biblioteca de Peptídeos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de Complemento/metabolismo , Alanina/metabolismo , Antígenos CD/genética , Antígenos CD/isolamento & purificação , Ligação Competitiva , Linhagem Celular , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar , Expressão Gênica , Humanos , Mutação , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/isolamento & purificação , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Receptores de Complemento/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The anaphylatoxic peptide C5a is an important inflammatory mediator of the complement system. We have generated human C5a-receptor (hC5aR) mutants with truncation of its cytosolic carboxyl-terminus (C-terminus). Both mutants were analysed for C5a-binding in transiently expressing COS cells, and one mutant additionally for GTP-binding regulatory protein (G-protein) coupling in cRNA-injected Xenopus oocytes. Our data suggest that (a) amino acids (aa) 314 to 326 as part of the C-terminus are necessary for proper receptor folding or expression and (b) the receptor C-terminus distal from position 327 is not critical for receptor expression, folding, binding and G-protein coupling.
Assuntos
Complemento C5a/metabolismo , Oócitos/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Complemento/química , Transdução de Sinais/fisiologia , Animais , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Feminino , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica , Humanos , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Receptor da Anafilatoxina C5a , Receptores de Complemento/genética , Receptores de Complemento/fisiologia , Relação Estrutura-Atividade , Transfecção , Xenopus laevisRESUMO
The human leukocyte chemoattractant receptors for complement factor C5a (C5a-R) and N-formylated peptides (fMLF-R) are important members of the superfamily of G-protein coupled receptors (GPCR). Uniquely among the GPCR, these two receptors cannot be expressed in a functionally active form in the oocytes of the frog Xenopus laevis, but require substitution of total RNA of the myelomonocytic U-937 or HL-60 cell lines, respectively. Recently, it was reported that the C5a-R may couple to the alpha subunit of G-16. We have tested this G-protein for its ability to complement the signal transduction cascade of the C5a-R and fMLF-R in Xenopus oocytes. Injection of cRNA for the C5a-R in combination with G alpha-16 led to expression of a functional C5a-R as measured by ligand-induced whole cell current. In contrast to a previous report, the fMLF-R exhibited some residual functional activity when transiently expressed in Xenopus oocytes the extent of which could, however, substantially be increased by coexpression of G alpha-16. Thus, G alpha-16 complements the signal transduction cascade of both receptors in Xenopus laevis oocytes and is most likely the complementing factor present in the U-937 and HL-60 cell lines.
Assuntos
Antígenos CD/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores de Complemento/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Animais , Complemento C5a/farmacologia , Condutividade Elétrica , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Oócitos , Receptor da Anafilatoxina C5a , Receptores de Formil Peptídeo , Proteínas Recombinantes/metabolismo , XenopusRESUMO
cRNA from a PCR-generated C5aR clone was prepared by in vitro transcription and microinjected into Xenopus laevis oocytes. Ligand-induced whole cell current could be detected after co-injection of cRNA for the C5aR with total RNA of the unstimulated U937 cell line, but not with either of the components injected alone. These data clearly demonstrate an absolute requirement of the C5aR for an additional human factor to become functionally expressed in Xenopus oocytes.