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.

Los autores no tienen nada que revelar.

Con la disminución de las tierras agrícolas disponibles y el aumento de la población, los investigadores de todo el mundo tienen el reto con las necesidades apremiantes para el mejoramiento de cultivos. El rendimiento puede ser muy influenciada por diversos estreses bióticos y abióticos. Entre ellos, la infección por patógenos es una de las principales causas de la reducción del rendimiento de los cultivos, responsable de aproximadamente 12% las pérdidas sólo en los EE.UU. 45. Para resolver este pro...

Debido a su naturaleza sésiles, las plantas están constantemente amenazados por una gran cantidad de patógenos que presentan diferentes estilos de vida y estrategias nutricionales 1. En una primera aproximación, los patógenos biotróficos mantienen su huésped vivo para recuperar los nutrientes, mientras que los agentes patógenos necrotróficos toxinas activamente secretas y enzimas para matar tejido del huésped y se alimentan de las células muertas de la 1. Otro grupo de agentes patógenos, hemibiotrophs denominados, comienza su curso con la etapa de la infección biotrófico y los cambios a la etapa necrotrófico al alcanzar un cierto umbral de acumulación patógeno 2. Con el fin de defender eficazmente contra estos microorganismos, las plantas han desarrollado un sistema inmune innato complicada equipado con múltiples mecanismos de vigilancia para detectar el ataque de patógenos y desencadenar la muerte celular localizada 3, así como resistencia sistémica adquirida (SAR) 4 programado. La investigación actual se centra en la caracterización de la sig esencialcomponentes naling y cruzadas conversaciones dentro del sistema inmunológico planta 5. Tal como se propone en el modelo de &quot;Zig-Zag&quot; 5, la primera capa de la respuesta inmune innata planta requiere la presencia de la membrana plasmática-localizada Patrón receptores de reconocimiento (PRRS) para detectar la invasión de un microbio. Derechos y responsabilidades parentales son capaces de reconocer patrones Microbio asociada Moleculares (mAmps) y establecer Inmunidad-Triggered MAMP (MTI) 6. Además de inducir una regulación positiva de la transcripción de genes que codifican proteínas PR antimicrobianos 7, MTI conduce a una variedad de eventos que la detención del crecimiento de patógenos, incluyendo la producción de especies reactivas del oxígeno (ROS) y reactivas de nitrógeno Especies (RNS), deposición de callose a la pared celular así como la activación de la señalización de vías múltiples quinasa 8. Hasta ahora, varios MAMPs se han identificado para desencadenar MTI en Arabidopsis, incluyendo flg22 bacteriana 9 </sup&gt; (Un fragmento de ácido 22 amino derivado de flagelina), elf18 10 (18 aminoácidos del factor de elongación de la traducción bacteriana Tu) y un componente estructural de la pared celular peptidoglicanos 11. Para establecer una infección exitosa, algunos patógenos especializados han desarrollado la capacidad de las proteínas efectoras secretas virulencia en los espacios intercelulares o intracelulares, y en consecuencia reprimir MTI y desencadenar Susceptibilidad-Triggered efectoras (ETS) 12,13. Por ejemplo, efectores de virulencia pueden inactivar proteína activada por mitógeno cascadas de quinasas (MAPK) fosforilación de MTI para inducir el desarrollo de la enfermedad dentro del tejido infectado 14-16 de. Durante la co-evolución dinámica entre los ejércitos y agentes patógenos, las plantas también desarrollaron la estrategia de contraataque para reconocer las proteínas efectoras y atenuar la virulencia de patógenos moléculas 17. Este reconocimiento directo o indirecto efector está mediada por resistencia a enfermedades (R) 18 proteínas. La mayoría de tdobladillo son miembros de NB-LRR (nucleótidos vinculante y ricos en leucina repite) la familia 19. La percepción de un efector no virulenta por una proteína R provoca una respuesta más fuerte y más amplia inmunológico caracterizado como Inmunidad Accionado efector (ETI) 20. Además de inducir la expresión de genes de defensa 21 y la producción de metabolitos de defensa 22, ETI menudo conduce a una localizada muerte celular programada conocida como hipersensible rápida respuesta (HR) para restringir el patógeno se propague en el tejido adyacente 3. Además de la muerte celular programada localizada 23, las plantas son capaces de iniciar un largo plazo y la respuesta inmune en todo el sistema denominado Systemic Acquired Resistencia (SAR) 4. Tras la estimulación con un patógeno biotrófico, las células vegetales desencadenan la biosíntesis y acumulación de un ácido fitohormona endógena salicílico (SA) y proteínas PR en ambos tejidos locales y sistémicos 24. A través de tsu proceso, se logra un mayor estado de preparación en las hojas infectadas que permite para el montaje de las respuestas de defensa más rápidos durante una infección posterior por un amplio espectro de patógenos 24. SA y sus análogos sintéticos como el benzo (1,2,3), 7-carbotioico tiadiazol éster del ácido S metil (BTH) y ácido 2,6-dicloroisonicotínico (INA) son capaces de inducir químicamente ácido salicílico (SA) Inmunidad -Accionados (ITS) tras la aplicación externa de 24. Nonexpressor de los genes relacionados con la patogénesis (1 NPR1) se propone para ser uno de los receptores SA y funciona como un importante regulador transcripcional durante la respuesta de defensa mediada por SA en ambos tejidos locales y sistémicos 21,25,26. Se ha demostrado de manera concluyente que NPR1 se requiere para el establecimiento SAR y la pérdida de NPR1 conduce a la susceptibilidad dramática hacia Pseudomonas syringae 25. Para caracterizar ampliamente la contribución molecular de plantascomponentes en las interacciones planta-patógeno, múltiples bioensayos se han desarrollado para medir acontecimientos de defensa específicos, incluyendo ROS estalló 27, deposición callose 28, genes de defensa expresión y acumulación de sus productos proteicos 21. Si bien estos ensayos individuales pueden proporcionar información en una forma específica de la respuesta inmune de la planta, ninguno de ellos, sin embargo, son capaces de representar la respuesta de defensa completa sobre todo el nivel de la planta. Por el contrario, la cuantificación del crecimiento de patógenos después de la infección proporciona una estimación global de la respuesta inmune a nivel del organismo. Por lo tanto, el desarrollo y optimización de un ensayo de inoculación de patógenos preciso y altamente estandarizado es crítico para alimentar la investigación y descubrimientos en las respuestas inmunes de Arabidopsis. Pseudomonas syringae, una bacteria Gram-negativa, se identificó como un fitopatógeno capaz de causar enfermedad en una gama de huéspedes vegetales incluyendo Arabidopses 29. A medida que el sistema de la planta de patógenos modelo, la Arabidopsis - P. interacción syringae se ha aplicado ampliamente para comprender los mecanismos moleculares respuestas de defensa de las plantas subyacentes 29. Hasta ahora, más de 50 P. patovares syringae se han identificado basándose en su capacidad para infectar diferentes especies de plantas 30 . P. syringae pv. DC3000 tomate (Pst DC3000) 31 y P. syringae pv. maculicola ES4326 (PSM ES4326) 32 son las dos cepas virulentas más usados ​​y ampliamente caracterizados. Aparte de ser reconocido por la planta y la activación de la respuesta de MTI, Pst DC3000 y PSM ES4326 son capaces de secretar proteínas efectoras virulentas para suprimir MTI y desencadenar ETS para favorecer el 31,33 crecimiento de patógenos. Para diseccionar funcionalmente la interacción entre Arabidopsis y P. syringae, múltiple0; métodos de infección de patógenos se han desarrollado sobre la base del enfoque de prestación de patógenos. Para las plantas de suelos cultivados, patógeno puede ser entregado por la infiltración de la jeringa, la infiltración de vacío, la inoculación por inmersión y la inoculación aerosol 29,34. Recientemente, ensayo de inundación de la inoculación de las plántulas fue desarrollado para realizar pantallas de gran escala en el cultivo de tejidos placas cultivadas plantas de Arabidopsis jóvenes 35. Infiltración Jeringa, como uno de los método más comúnmente utilizado, entrega manualmente el patógeno en el apoplasto través de las aberturas de las hojas naturales estomas 29 llamados. A través de este enfoque, la misma cantidad de P. syringae se puede infiltrar en la hoja infectada y la fuerza de la respuesta inmune planta está inversamente correlacionada con los niveles de crecimiento de patógenos. Por lo tanto, la cuantificación del crecimiento de patógenos sirve como un enfoque óptimo para evaluar la función inmune a nivel de planta entera. Además, la infiltración de la jeringa puede distinguir el tejido local y sistémica, que puede be aplicable en la caracterización de los mecanismos moleculares que subyacen a SAR 36. En el siguiente protocolo, se describe un ensayo de infiltración jeringa optimizado con PSM ES4326 para detectar mutantes de Arabidopsis para una mayor susceptibilidad a la enfermedad (EDS). Este protocolo empleará dos genotipos de Arabidopsis: una de tipo salvaje ecotipo Columbia-0 (Col-0) plantas (de control) y npr1-1 mutantes de pérdida de función (hipersusceptibles) que se infectaron con la cepa bacteriana virulenta PSM ES4326 37. El mutante npr1-1 lleva una mutación puntual dentro de la secuencia de consenso-ankyrin repetida de la molécula NPR1, que altera la histidina altamente conservada en tirosina y hace que la proteína no funcional 25. Además, una serie de modificaciones del ensayo de infiltración de la jeringa se describen que permiten la cuantificación de defectos en las capas específicas de la respuesta inmune, incluyendo MTI y STI.

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

En ausencia de las células inmunes móviles especializadas, las plantas utilizan su localizada muerte celular programada y sistémica resistencia adquirida a defenderse contra el ataque de patógenos. La contribución de un gen de Arabidopsis específica a la respuesta inmune general de la planta puede ser específicamente y cuantitativamente evaluado ensayando el crecimiento de patógenos en el tejido infectado. Durante más de tres décadas, la bacteria hemibiotrophic Pseudomonas syringae pv. Maculicola ES4326 (PSM ES4326) se ha aplicado ampliamente como el modelo de patógenos para investigar los mecanismos moleculares que subyacen a la respuesta inmune de Arabidopsis. Para entregar agentes patógenos en el tejido de la hoja, múltiples métodos de inoculación se han establecido, por ejemplo, infiltración de la jeringa, la inoculación por inmersión, pulverización, infiltración al vacío, y la inoculación de inundación. El siguiente protocolo describe un método de infiltración jeringa optimizado para ofrecer virulenta PSM ES4326 en hojas de adultossuelo cultivado plantas de Arabidopsis y la pantalla con precisión para una mayor susceptibilidad a la enfermedad (EDS) en dirección a este patógeno. Además, este protocolo se puede complementar con múltiples tratamientos previos para diseccionar más defectos inmunes específicas dentro de las diferentes capas de defensa de la planta, incluyendo el ácido salicílico (SA) Inmunidad -Accionados (ITS) e Inmunidad-Triggered MAMP (MTI).

El protocolo que describimos aquí representa un P. optimizado syringae ensayo de infiltración de jeringa para evaluar cuantitativamente la respuesta inmune en plantas de Arabidopsis. Como se ilustra en la Figura 1, la infiltración jeringa de PSM ES4326 es seguido por la extracción y cuantificación de patógenos a través de diluciones seriadas y colonias enumeración. Como se describe en el Paso 3 en el texto del prot...

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.

Ensayo de infiltración bacteriana de la hoja por Fine Caracterización de las respuestas de defensa de la planta utilizando el<em&gt; Thaliana-Pseudomonas syringae Arabidopsis</em&gt; Patosistema

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

SCIENTISTS IN ACTION

21.5K vistas.  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.

Video: 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.

Ensayo para asociados a patógenos patrón molecular (PAMP)-Accionados Inmunidad (PTI) en las plantas

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

Investigación

Inmunología e Infección

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.

Evaluación de la respuesta estomática de las células bacterianas en vivo utilizando imágenes de Hoja Entera

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.

Asistida por microondas diagnóstico rápido de enfermedades de virus de plantas por microscopía electrónica de transmisión

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.

La medición de la carga bacteriana y la respuesta inmune en ratones infectados con 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.

La visualización de las respuestas inducidas por toxinas bacterianas mediante microscopía de fluorescencia de células en vivo

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.

La infección bacteriana sistémica y Defensa Inmune fenotipos en<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.

Aplicación de Ensayo inmunoensayo de largo plazo culta Interferón-γ ligado a enzimas para la evaluación efectoras y de memoria T respuestas celulares en el ganado

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.

Caracterización de las respuestas de isotipo de inmunoglobulina dependiente del timo y timo-independiente en ratones usando el análisis enzima-ligado del inmunosorbente

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 un patógeno bacteriano a punta de prueba para las respuestas celular y organísmica nivel Host

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.

Opsonofagocítico asesinato ensayo para evaluar las respuestas inmunológicas 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 ...