We thank Dr. Shahid Mukhtar for critiquing the manuscript and Dr. Xinnian Dong for the sample data analysis file. This work is supported by a NSF-CAREER award (IOS-1350244) to KPM and the UAB Biology Department.

Os autores não têm nada a revelar.

Com a diminuição da terra disponível e aumento da população, os investigadores de todo o mundo são desafiados com as necessidades prementes para melhoramento de culturas. O rendimento pode ser muito influenciado por vários estresses bióticos e abióticos. Entre eles, a infecção por patógeno é uma das principais causas de redução do rendimento das culturas, responsável por aproximadamente 12% as perdas em os EUA sozinhos 45. Para resolver este problema, a pesquisa maciça tem sido conduzida no m...

Devido à sua natureza sessile, as plantas são constantemente ameaçados por uma infinidade de agentes patogénicos que apresentam vários estilos de vida e estratégias nutricionais 1. Para uma primeira aproximação, patógenos biotróficos manter seu hospedeiro vivo para recuperar nutrientes, enquanto patógenos necrotróficos toxinas e enzimas ativamente secretas para matar o tecido hospedeiro e se alimentam de células mortas 1. Outro grupo de patógenos, denominados hemibiotrophs, começa seu curso de infecção com o estágio biotrófica e mudanças para o palco necrotrophic ao atingir um determinado limiar de acumulação patógeno 2. A fim de defender-se eficazmente contra esses microorganismos, plantas desenvolveram um sistema imunológico inato complicado equipado com múltiplos mecanismos de vigilância para detectar o ataque de patógenos e desencadear localizada a morte celular programada 3, bem como a resistência adquirida sistémica (SAR) 4. A pesquisa atual está focada em caracterizando o sig essencialnaling componentes e transversais conversações dentro do sistema imunológico da planta 5. Tal como proposto no modelo de &quot;ziguezague&quot; 5, a primeira camada da resposta imune inata planta requer a presença de plasma-membrana localizada Reconhecimento de Padrões Receptores (SRRS) para detectar a invasão de um micróbio. PRRs são capazes de reconhecer padrões Microbe-Associated Molecular (mAmps) e estabelecer Imunidade MAMP-Provocado (MTI) 6. Além de induzir uma regulação positiva da transcrição de genes que codificam proteínas PR antimicrobianos 7, MTI leva a uma variedade de eventos que parar o crescimento de agentes patogénicos, incluindo a produção de espécies de oxigénio reactivos (ROS) e reactiva de azoto Espécie (RNS), deposição de calose à parede celular , bem como a activação de múltiplas vias de sinalização da cinase 8. Até agora, vários mAmps foram identificados para desencadear MTI em Arabidopsis, incluindo flg22 bacteriana 9 </sup&gt; (Um fragmento de 22 aminoácidos derivado de flagelina), 10 (18 elf18 aminoácidos do factor de tradução bacteriana alongamento Tu) e um componente da parede celular estrutural 11 peptidoglicanos. Para estabelecer uma infecção bem sucedida, alguns patógenos especializados têm evoluído a capacidade de proteínas efetoras secretos virulência para os espaços intracelulares ou intercelulares e, consequentemente, reprimir MTI e desencadear Suscetibilidade Effector-Provocado (ETS) 12,13. Por exemplo, efectores de virulência pode inactivar Mitogen-Activated Protein Kinase cascatas (MAPK) de fosforilação de MTI para induzir o desenvolvimento da doença no tecido infectado 14-16. Durante a co-evolução dinâmica entre hospedeiros e patógenos, as plantas também desenvolveu a estratégia de contra-ataque para reconhecer as proteínas efetoras e atenuar a virulência de patógenos moléculas 17. Este reconhecimento direto ou indireto efetora é mediada por proteínas de resistência a doenças (R) 18. Mais de them são membros do NB-LRR (Nucleotide Binding e leucina-ricos repetições) da família 19. A percepção de um efetor avirulent por uma proteína R provoca uma resposta imune mais forte e mais amplo caracterizada como Imunidade Effector-Provocado (ETI) 20. Além de induzir a expressão de genes de defesa 21 e a produção de metabolitos de defesa 22, ETI muitas vezes leva a uma rápida morte celular programada localizada conhecida como resposta hipersensível (HR) para restringir o agente patogénico a partir de espalhamento para o tecido adjacente 3. Além da morte celular programada localizada 23, as plantas são capazes de iniciar um a longo prazo e a resposta imunitária global do sistema denominado a resistência adquirida sistémica (SAR) 4. Após o desafio com um agente patogénico biotrofica, células vegetais desencadear a biossíntese e acumulação de um ácido salicílico fitohormônio endógena (SA) e proteínas PR em ambos os tecidos locais e sistémicos 24. Através de tseu processo, um elevado estado de prontidão é alcançado nas folhas não infectados que permite a montagem respostas de defesa mais rápidos durante uma infecção subsequente por um amplo espectro de agentes patogénicos 24. SA e os seus análogos sintéticos, tais como o benzo (1,2,3) -tiadiazol-7-carbotióico éster do ácido S-metil (BTH) e ácido 2,6-dicloroisonicotínico (INA) são capazes de induzir quimicamente ácido salicílico (AS) Imunidade Triggered (STI) mediante aplicação externa 24. Nonexpressor de genes relacionados-Patogênese 1 (NPR1) se propõe a ser um dos receptores e funções SA como principal regulador da transcrição durante a resposta de defesa SA-mediada em ambos os tecidos locais e sistêmicos 21,25,26. Tem sido demonstrado de forma conclusiva que NPR1 é necessária para a criação de SAR e a perda de NPR1 conduz a susceptibilidade dramática no sentido 25 de Pseudomonas syringae. Para caracterizar a contribuição extensivamente molecular de plantacomponentes nas interacções planta-patógeno, vários bioensaios foram desenvolvidos para medir a eventos específicos de defesa, incluindo a explosão de ROS 27, 28 deposição de calose, expressão dos genes de defesa e acumulação de produtos de proteína de 21 suas. Embora estes ensaios individuais pode fornecer informações para uma forma específica da resposta imune da planta, nenhum deles, no entanto, são capazes de representar a resposta de defesa completa em toda a nível da planta. Por outro lado, a quantificação do crescimento do patógeno após a infecção fornece uma estimativa global da resposta imunitária ao nível do organismo. Portanto, o desenvolvimento e otimização de um ensaio de inoculação do patógeno preciso e altamente padronizada é fundamental para abastecer-se a pesquisa e descobertas sobre as respostas imunes de Arabidopsis. Pseudomonas syringae, uma bactéria Gram-negativa, foi identificado como um fitopatógeno capaz de causar doença em uma gama de espécies de plantas, incluindo Arabidopsé 29. Como o modelo do sistema planta-patógeno, a Arabidopsis - P. interação syringae tem sido amplamente aplicada para compreender os mecanismos moleculares subjacentes respostas de defesa da planta 29. Até agora, mais de 50 P. patovares syringae foram identificadas com base em sua capacidade de infectar diferentes espécies de plantas 30 . P. syringae pv. DC3000 tomate (Pst DC3000) 31 e P. syringae pv. maculicola ES4326 (ES4326 Psm) 32 são as duas estirpes virulentas mais utilizados e extensivamente caracterizados. Além de ser reconhecido pela planta e desencadeamento da resposta MTI, Pst DC3000 e Psm ES4326 são capazes de segregar proteínas efetoras virulentos para suprimir MTI e desencadear ETS para favorecer o crescimento de patógenos 31,33. Para dissecar funcionalmente a interação entre Arabidopsis e P. syringae, múltiplos0; infecção patogénica métodos foram desenvolvidos com base na abordagem de entrega de agente patogénico. Para as plantas cultivadas no solo, patógeno pode ser entregue por infiltração seringa, infiltração de vácuo, a inoculação mergulho e inoculação de pulverização 29,34. Recentemente, ensaio inundação-inoculação de mudas foi desenvolvido para executar telas em grande escala em placas de cultura de tecidos cultivados jovens plantas de Arabidopsis 35. Seringa de infiltração, como um dos abordagem mais utilizada, proporciona manualmente o agente patogénico para o apoplasto através das aberturas de folhas naturais chamados estomas 29. Através desta abordagem, quantidades iguais de P. syringae pode ser infiltrada na folha infectada e a força da resposta imune da planta está inversamente correlacionada com os níveis de crescimento do patógeno. Portanto, a quantificação do crescimento do patógeno serve como uma melhor abordagem para avaliar a função imune a todo o nível da planta. Além disso, a seringa pode distinguir a infiltração do tecido local e sistémica, a qual pode be aplicável em caracterizar os mecanismos moleculares subjacentes SAR 36. No seguinte protocolo, descrevemos um ensaio infiltração seringa otimizado com Psm ES4326 para rastrear mutantes de Arabidopsis para maior susceptibilidade à doença (EDS). Este protocolo irá empregar dois genótipos de Arabidopsis: a de tipo selvagem ecótipo Columbia-0 (Col-0) plantas (controle) e npr1-1 mutantes de perda de função (hypersusceptible) que será infectada com a cepa bacteriana virulenta Psm ES4326 37. O mutante npr1-1 transporta uma mutação pontual no interior da sequência de consenso de anquirina-repetição da molécula NPR1, o que altera a histidina altamente conservado e a tirosina torna a proteína não funcional 25. Além disso, um certo número de modificações do ensaio de seringa infiltração estão descritos que permitem a quantificação de defeitos nas camadas específicas da resposta imunitária, incluindo MTI e STI.

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bacterial leaf infiltration assay for fine characterization of plant defense responses using the arabidopsis thaliana-pseudomonas syringae pathosystem

Na ausência de células imunes celulares especializadas, as plantas utilizam sua localizada morte celular programada e sistêmica a resistência adquirida para se defender contra o ataque de patógenos. A contribuição de um gene Arabidopsis específico para a resposta imunitária geral da planta podem ser especificamente e quantitativamente avaliado por análise do crescimento do patógeno no tecido infectado. Por mais de três décadas, a bactéria Pseudomonas syringae pv hemibiotrophic. Maculicola ES4326 (ES4326 Psm) tem sido amplamente aplicada como modelo patógeno para investigar os mecanismos moleculares subjacentes a resposta imune Arabidopsis. Para entregar patógenos para o tecido foliar, foram estabelecidos vários métodos de inoculação, por exemplo, a infiltração seringa, inoculação mergulho, spray, infiltração a vácuo, e inoculação de inundação. O protocolo a seguir descreve um método de seringa infiltração otimizada para oferecer virulenta Psm ES4326 em folhas de adultosolo-grown plantas de Arabidopsis e rastrear com precisão para maior susceptibilidade à doença (EDS) em direção a este patógeno. Além disso, este protocolo pode ser complementado com vários pré-tratamentos para dissecar ainda mais defeitos imunes específicos dentro de diferentes camadas de defesa vegetal, incluindo ácido salicílico (SA) Imunidade Triggered (STI) e Imunidade MAMP-Provocado (MTI).

O protocolo aqui descrito representa um P. otimizado syringae ensaio seringa infiltração para avaliar quantitativamente a resposta imune em plantas de Arabidopsis. Tal como ilustrado na Figura 1, a seringa infiltração do PSM ES4326 é seguido por extracção e quantificação patogénio através de diluições em série e as colónias enumeração. Tal como descrito no Passo 3 dentro do texto do protocolo, susceptibili...

Watch this Scientific Journal Video about Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem at JoVE

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem | Immunology and Infection | Video

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Bacteriana Folha Infiltração Ensaio para Belas Caracterização das respostas de defesa da planta usando o Arabidopsis thaliana-Pseudomonas syringae Patossistema

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

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Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Plant Biology

Plant Responses to the Environment

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21.5K visualizações.  University of Alabama at Birmingham. Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

Vídeo: Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

Ensaio para Pathogen-Associated Padrão Molecular (PAMP) Triggered-Imunidade (PTI) em Plantas

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

Imunologia e Infecção

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Avaliar a resposta estomática ao vivo células bacterianas usando imagens Folha Inteira

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Microondas Assistida Diagnóstico Rápido de Doenças vírus de planta por Microscopia Eletrônica de Transmissão

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Medir a carga bacteriana e resposta imune em camundongos infectados com Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Visualização das respostas toxina bacteriana induzida por microscopia de fluorescência de células vivas

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

Infecção bacteriana sistêmica e Imune Defesa Phenotypes em<em&gt; Drosophila melanogaster</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Aplicação de longo prazo cultivadas Interferon-γ Enzyme-linked Immunospot Ensaio para a Avaliação e efetoras de memória T respostas de células em gado

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Caracterização das respostas de isotipo de imunoglobulina Timo-dependentes e Timo-independente nos ratos usando ensaio enzima-lig da imunoabsorção

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Usando um patógeno bacteriano para sonda para celular e organísmica-nível Host respostas

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Ensaio de matar Opsonophagocytic para avaliar as respostas imunológicas contra patógenos bacterianos

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...