Los autores agradecen a Therwa Hamza, John E. Tidwell, Clovis Nina, y Suzanne Smith para la asistencia experimental y Suzanne Smith para corrección de pruebas. Los autores también agradecen a John Thomas, PhD para la prestación de los aislados bacterianos clínicos y John B. Barnett, PhD, por su apoyo y el uso del laboratorio de seguridad biológica en el Departamento de Microbiología, Inmunología y Biología Celular en la Universidad de Virginia Occidental. Los autores agradecen el apoyo financiero de la Osteosíntesis y Trauma Care Foundation y la National Science Foundation (# 1003907). Microscopio experimentos y análisis de imagen se realizaron también en el West Virginia University Fondo Imaging, que es apoyado en parte por el Mary Babb Randolph Cancer Center y el NIH subvención P20 RR016440. El uso de animales para la extracción de sangre fueron aprobados por el Cuidado de Animales Universidad West Virginia institucional y el empleo. Todos los experimentos fueron ejecutadas en conformidad con todas las guidelin relevanteEs, regulaciones y agencias reguladoras.

1. Preparación y activación de PRP 1.1 Extracción de sangre <ol><li> Anestesiar conejo por inhalación de isoflurano (2% en O 2 para la inducción y 1% para el mantenimiento). </li><li> Dibujar 2 ml 0,129 M citrato trisódico (una solución anticoagulante) en una jeringa de 20 ml. La solución de citrato tri-sódico se prepara disolviendo 1,897 g de tri-citrato de sodio en 50 ml de H 2 O destilada y filtrar con un filtro de 0,22 micras estéril. </li><li> Esterilizar la oreja del conejo utilizando 70% de etanol. </li><li> Extraer la sangre (por ejemplo, 5 ml) de la vena de la oreja de conejo a través de una aguja de mariposa (25 G) conectado a la jeringa. </li><li> Mezclar la sangre con la solución de citrato de tri-sodio por agitación suave. La relación de volumen de sangre y tri-sodio solución de citrato es de 9:1. </li></ol> 1,2 PRP preparación (Figura 1) <ol><li> Transferir la sangre anticoagulada a un tubo de 50 ml de centrífuga de plástico. Tomar una alícuota de 10 lde sangre para determinar el recuento de plaquetas basal utilizando hemocitometría. </li><li> Se centrifuga la sangre a 300 xg durante 10 min a temperatura ambiente (RT) en una centrífuga con un rotor basculante. Establecer la aceleración y la velocidad de frenado a baja (Figura 1). </li><li> Después de la centrifugación, la sangre se separa en tres capas. La capa inferior es principalmente células de la sangre rojas, la capa media (comúnmente conocido como la &quot;capa leucocitaria&quot;) se compone de concentrados de plaquetas y leucocitos, y la capa superior es principalmente plasma, que es el componente líquido de la sangre, y las plaquetas ( Figura 1). Transportar con cuidado el tubo de centrífuga a una campana de cultivo de células; no molestar las capas. Transferir todo el plasma, capa leucocitaria, y 2-3 mm de espesor capa de glóbulos rojos en un tubo de plástico de 15 ml usando una pipeta de 1 ml de plástico. </li><li> Centrifugar la muestra transferida por segunda vez a 3.000 xg durante 15 min a TA. La capa superior (sobrenadante) se considera plasma pobre en plaquetas (PPP) und es transferido a un nuevo tubo. </li><li> Obtener PRP mediante el ajuste de la concentración de plaquetas en la muestra de sangre restante utilizando PPP para obtener 2,0 x 10 6 plaquetas / l (determinado por hemocitometría). </li></ol> 1,3 PRP activación <ol><li> Preparar la solución de PRP activación mediante la disolución de 5.000 IU trombina bovina con 5 ml de cloruro de calcio 10% a la concentración de trabajo de 1.000 UI / ml. </li><li> Añadir la solución de activación de PRP y PPP, y mezcle la solución pipetear repetidamente para formar PRP y PPP-geles. La relación en volumen de la solución de activación de PRP o PPP es de 1:4. </li></ol> 2. Prueba in vitro a los antimicrobianos de PRP usando el ensayo de Kill curva (Figura 2) <ol><li> El uso de un asa de inoculación estéril, añadir varias colonias de S. aureus a partir de su cultivo en placa durante la noche en 5 ml de caldo Mueller Hinton (MHB) en un tubo de plástico. Vórtice brevemente y luego incubar la muestra durante 2 horas a 37 ° C. Próximo, La densidad óptica de los medios de comunicación bacteriana se determinó utilizando un espectrofotómetro y se ajustó a una densidad óptica igual a ~ 1 x 10 8 UFC / ml en base a la curva estándar predeterminado. </li><li> Hacer una dilución de 100 veces utilizando PBS para obtener 1 x 10 6 UFC / ml y colocar el inóculo en hielo. </li><li> Configurar y etiquetar estériles, desechables 5 ml de fondo redondo tubos de poliestireno, y preparar los siguientes grupos de la muestra como se indica en la Tabla 1 para un volumen final de 2 ml en cada tubo. </li><li> Añadir PRP, PPP, o PBS primero a los tubos de poliestireno, seguido por la solución de trombina para la activación (formación de gel). A continuación, agregue MHB y luego la S. inóculos aureus (1 x 10 6 UFC / ml) para obtener la concentración final de 1 x 10 5 UFC / ml. </li><li> Incubar los tubos a 37 ° C con agitación orbital a 150 rpm. </li><li> En los puntos de tiempo predeterminados (por ejemplo, 0, 1, y h 2), mezclar las soluciones en cada tubo mediante pipeteo repetido (estepaso es importante ya que las bacterias pueden ser atrapados dentro del gel de PRP). Tomar 10 l de muestra, diluir en serie con una solución salina estéril al 0,9%, y pipetear una parte alícuota de 100 l de cada dilución en placas en un agar de tripticasa de soja (TSA, con sangre de oveja al 5%) para el recuento de CFU placa. </li><li> Cultura las placas de agar durante la noche a 37 º C, y luego contar y registrar las colonias de las placas. Representan los datos en una escala logarithimic con el tiempo (hr) en el eje X y UFC / ml en el eje y. </li></ol>

<ol>
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Los autores declaran que no tienen intereses en conflicto financieros.

Plasma rico en plaquetas se ha usado cada vez más para aplicaciones clínicas debido a sus propiedades de promoción de la cicatrización 15-17. En el presente estudio, el PRP se presenta como un nuevo enfoque para la prevención de infecciones. PRP se encontró que tienen fuertes propiedades antimicrobianas contra MRSA, MSSA, estreptococo del grupo A, y Neisseria gonorrhoeae. Las principales ventajas de PRP, en comparación con los tratamientos convencionales con antibióticos para la prev...

Implante asociada a la infección es una complicación clínica significativa. Staphylococcus aureus (S. aureus) es uno de los microorganismos más comunes aislados de infecciones relacionados con el implante. Es capaz de producir una biopelícula que recubre las superficies de implantes y puede conducir a 1,2 infección resistente a antibióticos. El tratamiento de las infecciones asociadas a implantes con frecuencia requiere hospitalización a largo plazo para desbridamientos repetidos y prolongados de terapia antibiótica parenteral. En los casos resistentes a antibióticos, la extracción del implante puede ser necesario. La creciente resistencia de las bacterias a los antibióticos también se ha referido a los Centros para el Control y Prevención de Enfermedades (CDC) como &quot;uno de los problemas de salud del mundo más urgentes.&quot; Con el tiempo, sin el desarrollo de tratamientos antimicrobianos nuevos y eficaces, es posible que los fármacos múltiples patógenos resistentes será intratable con los antibióticos convencionales. Prevención de implante asociadoinfección es por lo tanto importante y nuevos agentes profilácticos o enfoques son necesarios para la prevención de tales infecciones. Plasma rico en plaquetas (PRP) es una concentración de sangre autóloga que contiene más de 30 factores de crecimiento que pueden ayudar con la curación del hueso y de injerto de hueso 3-5. La aplicación de PRP para la reconstrucción ósea y maduración del tejido blando se ha informa cada vez más en las clínicas debido a su alta concentración de diversos factores de crecimiento liberados por las plaquetas. Varias características de PRP PRP indican que también pueden tener propiedades antimicrobianas 6-9. PRP contiene un gran número de plaquetas, una alta concentración de leucocitos (que puede poseer defensa del huésped-acciones contra bacterias y hongos), y varios péptidos antimicrobianos 7,8,10. En un estudio reciente de una gran cohorte de pacientes quirúrgicos cardíacos, se reveló que el uso intraoperatorio de PRP-gel durante el cierre de la herida significativamente disminuyó la incidencia de infección esternón superficial y profunda 11. Por estas razones y observaciones, la hipótesis de que el PRP, además de sus bien estudiadas promueven la curación de las propiedades, tiene propiedades antimicrobianas. Las ventajas potenciales de la utilización de PRP para prevenir la infección pueden incluir: (i) PRP es menos probable que induzca resistencia en comparación con los tratamientos convencionales con antibióticos. (Ii) PRP también tiene propiedades que favorecen la cicatrización que puede tener un efecto sinérgico en la prevención de infecciones; curación promotoras de PRP de propiedades podrían proporcionar un sello para evitar la adhesión bacteriana reduciendo así las probabilidades de infección por los patógenos y las células humanas están compitiendo para superficies de implante 12 , 13. (Iii) PRP es inherentemente biocompatible, seguro y libre de riesgo de las enfermedades transmisibles. Nuestro objetivo a largo plazo es el uso de PRP como un nuevo enfoque para prevenir implante asociada infections. El objetivo de este estudio fue la preparación de PRP utilizando un enfoque de centrifugación dos veces, para examinar PRP en propiedades antimicrobianas in vitro, y para describir los protocolos para evaluar tales propiedades antimicrobianas.

<table border="1">
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 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
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 <td>Bovine thrombin</td>
 <td>King Pharmaceuticals, Inc </td>
 <td>60793-215-05</td>
 <td>Thrombin (bovine origin)</td>
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 <td>Calcium chloride</td>
 <td>King Pharmaceuticals, Inc</td>
 <td>60793-215-05</td>
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 <td>E7023</td>
 <td></td>
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 <td>Baxter</td>
 <td>1001936060</td>
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 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
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 <td>Sigma-Aldrich</td>
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 <td></td>
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 <td>Sigma-Aldrich</td>
 <td>W302600</td>
 <td></td>
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 <td>Tryptic soy agar</td>
 <td>Fisher Scientific</td>
 <td>R01202</td>
 <td></td>
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 <td>Centrifuge</td>
 <td>Kendro Laboratory Products</td>
 <td>750043077</td>
 <td></td>
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 <td>Syringe filter</td>
 <td>Millipore</td>
 <td>SLGP033RS</td>
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prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

Implante infección asociada se está volviendo más y más difícil para la industria de la salud en todo el mundo debido a la creciente resistencia a los antibióticos, la transmisión de bacterias resistentes a antibióticos entre animales y seres humanos, y el alto costo de tratamiento de las infecciones. En este estudio, se describen una nueva estrategia que puede ser eficaz en la prevención de las infecciones relacionadas con implante basado en las propiedades antimicrobianas potenciales de plasma rico en plaquetas (PRP). Debido a sus propiedades bien estudiados para promover la curación, PRP (un producto biológico) ha sido cada vez más utilizado para aplicaciones clínicas, incluyendo cirugías ortopédicas, cirugía periodontal y oral, cirugías maxilofaciales, cirugías plásticas, medicina deportiva, etc PRP podría ser una alternativa avanzada a los tratamientos convencionales con antibióticos para prevenir las infecciones asociadas a implantes. El uso de PRP puede ser ventajoso en comparación con los tratamientos convencionales con antibióticos SINCE PRP es menos probable que induzca resistencia a los antibióticos y PRP antimicrobiana y propiedades curativas que promueven puede tener un efecto sinérgico en la prevención de infecciones. Es bien conocido que los agentes patógenos y las células humanas están compitiendo por superficies de los implantes, y PRP propiedades de curación promoción podría mejorar la unión celular humana reduciendo así las probabilidades de infección. Además, el PRP es inherentemente biocompatible, seguro y libre de riesgo de las enfermedades transmisibles. Para nuestro estudio, hemos seleccionado varias cepas clínicas de bacterias que se encuentran comúnmente en las infecciones ortopédicas y examinó si PRP tiene propiedades in vitro antimicrobianos contra estas bacterias. Hemos preparado PRP utilizando un enfoque de centrifugación dos veces, que permite la concentración de plaquetas mismo que debe obtenerse para todas las muestras. Hemos logrado resultados consistentes antimicrobianos y se ha encontrado que el PRP tiene fuerte propiedades in vitro antimicrobianos contra bacterias como methicillin sensible a la meticilina y resistentes a Staphylococcus aureus, Streptococcus del Grupo A, y Neisseria gonorrhoeae. Por lo tanto, la utilización de PRP puede tener el potencial para prevenir la infección y reducir la necesidad de un costoso tratamiento post-operatorio de infecciones relacionados con el implante.

PRP se preparó reproducible utilizando un enfoque de centrifugación dos veces (Figura 1). PRP se encuentra a presentar fuerte (hasta 100-veces la reducción de UFC) en las propiedades antimicrobianas in vitro contra S. aureus resistente aureus (MRSA) (Figura 3), que se encuentra comúnmente en los hospitales de todo el mundo 14. Del mismo modo, el PRP tiene fuertes propiedades antimicrobianas en contra de estafilococos sensibles S. au...

Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implante asociada a la infección es una complicación clínica significativa. Este estudio describe un enfoque utilizando plasma rico en plaquetas (PRP) para prevenir infecciones relacionados con el implante, presenta el protocolo para la preparación de PRP con concentración de plaquetas constante, e informa de las propiedades antimicrobianas de los recientemente identificados PRP y protocolos relacionados para examinar tales propiedades antimicrobianas<em&gt; In vitro.</em

PRP como un nuevo enfoque para prevenir la infección: Preparación y<em&gt; In vitro</em&gt; Propiedades antimicrobianas de PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

prp-as-new-approach-to-prevent-infection-preparation-vitro

Research

JoVE Journal

Immunology and Infection

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

21.2K vistas.  West Virginia University. Implante asociada a la infección es una complicación clínica significativa. Este estudio describe un enfoque utilizando plasma rico en plaquetas (PRP) para prevenir infecciones relacionados con el implante, presenta el protocolo para la preparación de PRP con concentración de plaquetas constante, e informa de las propiedades antimicrobianas de los recientemente identificados PRP y protocolos relacionados para examinar tales propiedades antimicrobianas In vitro...

Video: PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transducción retroviral de receptores de células T en ratones las células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Investigación

Inmunología e Infección

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

Simple y robusto En vivo Y In vitro Enfoque para el estudio de ensamblaje del virus

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de un medio artificial de esputo para probar la eficacia de antibióticos contra la<em&gt; Pseudomonas aeruginosa</em&gt; En condiciones más pertinentes a la fibrosis quística pulmonar

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

El uso de<em&gt; Galleria mellonella</em&gt; Como organismo modelo para estudiar<em&gt; Legionella pneumophila</em&gt; Infección

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Vigilar los cambios en la polaridad de la membrana, membrana Integridad, e intracelular de iones concentraciones en<em&gt; Streptococcus pneumoniae</em&gt; Uso de tintes fluorescentes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Un enfoque simple y eficiente de construcción de mutantes de virus vaccinia Vectores

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Retroviral transducción de células T colaboradoras como un enfoque genético para estudiar mecanismos que controlan su diferenciación y función

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...