Les auteurs remercient Therwa Hamza, John E. Tidwell, Nina Clovis, et Suzanne Smith pour son aide expérimentale et Suzanne Smith pour la relecture. Les auteurs remercient également Jean Thomas, Ph.D. de fournir les isolats bactériens cliniques et John B. Barnett, PhD, pour son soutien et à l&#39;utilisation du laboratoire de sécurité biologique au Département de microbiologie, d&#39;immunologie et de biologie cellulaire à l&#39;Université West Virginia. Les auteurs tiennent à remercier le soutien financier de l&#39;ostéosynthèse et Trauma Care Foundation et la National Science Foundation (# 1003907). Expériences de microscopie et analyse d&#39;images ont également été réalisés dans l&#39;installation de la West Virginia University Imaging, qui est soutenu en partie par le Mary Babb Randolph Cancer Center et NIH P20 RR016440. L&#39;utilisation d&#39;animaux pour les prises de sang ont été approuvés par les soins de la West Virginia University animaux institutionnel et Use Committee. Toutes les expériences ont été exécutées en conformité avec toutes les guidelin pertinentees, les règlements et les organismes de réglementation.

1. Préparation et activation du PRP 1,1 prélever du sang <ol><li> Anesthésier le lapin par inhalation d&#39;isoflurane (2% de O 2 pour l&#39;induction et 1% pour la maintenance). </li><li> Dessinez 2 ml 0,129 M citrate trisodique (une solution anticoagulant) dans une seringue de 20 ml. La solution de citrate trisodique est préparée par dissolution de 1,897 g de tri-sodium citrate dans 50 ml H 2 O distillée et filtrer avec un filtre stérile de 0,22 um. </li><li> Stériliser l&#39;oreille de lapin à l&#39;aide d&#39;éthanol à 70%. </li><li> Prélever du sang (par exemple 5 ml) à partir de la veine de l&#39;oreille de lapin par l&#39;intermédiaire d&#39;une aiguille à ailettes (25 G) connectée à la seringue. </li><li> Mélanger le sang avec la solution de citrate trisodique par agitation douce. Le rapport de volume de sang et de solution de citrate trisodique est de 9:1. </li></ol> 1.2 Préparation PRP (Figure 1) <ol><li> Transférer le sang coagulé dans un tube à centrifugation de 50 ml en plastique. Prélever une partie aliquote de 10 ulsang pour déterminer la numération plaquettaire de base en utilisant hemocytometry. </li><li> Centrifuger le sang à 300 xg pendant 10 min à température ambiante (TA) dans une centrifugeuse avec un rotor swing-out. Réglez l&#39;accélération et la vitesse de freinage à faible (Figure 1). </li><li> Après centrifugation, le sang est séparé en trois couches. La couche inférieure est de globules rouges principalement, la couche médiane (communément appelé le &quot;buffy coat&quot;) est composé de plaquettes concentrées et des leucocytes, et la couche supérieure est essentiellement du plasma, qui est le composant liquide du sang et des plaquettes ( Figure 1). Attentivement le transport du tube à centrifuger à une hotte de culture cellulaire; ne pas déranger les couches. Transférer la totalité du plasma, buffy coat, et 2-3 mm d&#39;épaisseur couche de globules rouges dans un tube de 15 ml en plastique à l&#39;aide d&#39;une pipette 1 ml en plastique. </li><li> Centrifuger l&#39;échantillon transféré une seconde fois à 3000 xg pendant 15 min à température ambiante. La couche supérieure (surnageant) est considéré plasma pauvre en plaquettes (PPP) d&#39;d est transférée dans un nouveau tube. </li><li> Obtenir PRP en ajustant la concentration de plaquettes dans l&#39;échantillon de sang restant en utilisant PPP pour obtenir 2,0 x 10 6 plaquettes / ul (déterminée par hemocytometry). </li></ol> 1.3 Activation du PRP <ol><li> Préparer la solution d&#39;activation PRP en dissolvant 5.000 thrombine bovine UI avec 5 ml de chlorure de calcium à 10% à la concentration de travail de 1000 UI / ml. </li><li> Ajouter la solution d&#39;activation de PRP et PPP, et mélanger la solution par pipetage à plusieurs reprises pour former PRP et PPP-gels. Le rapport en volume de la solution d&#39;activation de PRP ou PPP est de 1:4. </li></ol> 2. D&#39;essai in vitro aux antibiotiques des PRP Utilisation Assay Curve Kill (figure 2) <ol><li> En utilisant une boucle stérile inoculation, ajouter plusieurs colonies de S. aureus à partir de sa plaque de culture du jour au lendemain dans 5 ml de bouillon Mueller Hinton (MHB) dans un tube en plastique. Vortex brièvement, puis incuber l&#39;échantillon pendant 2 heures à 37 ° C. Suivant, La densité optique du support bactériennes a été déterminée à l&#39;aide d&#39;un spectrophotomètre et ajustées à une densité optique égale à environ 1 x 10 8 CFU / ml sur la base de la courbe étalon prédéterminée. </li><li> Faire une dilution 100x en utilisant du PBS pour obtenir 1 x 10 6 UFC / ml et placer les inoculums sur la glace. </li><li> Mettre en place et étiqueter stériles et jetables tubes de 5 ml en polystyrène à fond rond, et de préparer les groupes d&#39;échantillons suivants, comme indiqué dans le tableau 1 pour un volume final de 2 ml dans chaque tube. </li><li> Ajouter PRP, PPP, ou PBS premier des tubes de polystyrène, suivie de la solution de thrombine pour l&#39;activation (la formation du gel). Ensuite, ajoutez MHB puis le S. inoculums aureus (1 x 10 6 UFC / ml) pour obtenir la concentration finale de 1 x 10 5 UFC / ml. </li><li> Incuber les tubes à 37 ° C sous agitation orbitale à 150 rpm. </li><li> À des moments prédéterminés (par exemple 0, 1, et 2 h), mélanger les solutions dans chaque tube par pipetage répété (cetteétape est importante car les bactéries peuvent être piégés à l&#39;intérieur du gel PRP). Prenez 10 ul d&#39;échantillon, diluer en série avec une solution saline stérile à 0,9%, et introduire à la pipette une partie aliquote de 100 ul de chaque dilution sur une gélose trypticase soja (TSA, avec du sang de mouton à 5%) pour le comptage plaque UFC. </li><li> Culture des plaques de gélose pendant la nuit à 37 ° C, puis de compter et d&#39;enregistrer les colonies de la plaque. Tracer des données sur une échelle de temps logarithimic (h) sur l&#39;axe des x et UFC / ml sur l&#39;axe des ordonnées. </li></ol>

<ol>
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Les auteurs déclarent n&#39;avoir aucun conflit d&#39;intérêts financiers.

Plasma riche en plaquettes a été de plus en plus utilisées pour des applications cliniques en raison de ses propriétés favorisant la guérison 15-17. Dans la présente étude, le PRP a été présentée comme une nouvelle approche pour la prévention des infections. Le PRP a été constaté que de fortes propriétés antimicrobiennes contre le SARM, SASM, streptocoque du groupe A, et Neisseria gonorrhoeae. Les principaux avantages de la PRP, par rapport à des traitements antibiotiques ...

Implant associée à l&#39;infection est une complication clinique importante. Staphylococcus aureus (S. aureus) est l&#39;un des microorganismes les plus fréquemment isolés à partir implant infections nosocomiales. Il est capable de produire un biofilm qui recouvre les surfaces des implants et peut conduire à l&#39;infection de 1,2 résistante aux antibiotiques. Traitement des infections associées aux implants nécessite souvent une hospitalisation à long terme pour débridements répétés et prolongés thérapie parentérale d&#39;antibiotiques. En cas de résistance aux antibiotiques, le retrait de l&#39;implant peut être nécessaire. La résistance croissante des bactéries aux antibiotiques a également été évoquée par les Centers for Disease Control and Prevention (CDC) comme «l&#39;un des problèmes de la santé les plus pressants.&quot; Avec le temps, sans le développement de nouveaux traitements antimicrobiens et efficace, il est possible que plusieurs médicaments pathogènes résistants sera intraitable avec les antibiotiques classiques. Prévention de l&#39;implant associél&#39;infection est donc important et de nouveaux agents prophylactiques ou approches sont nécessaires pour prévenir ces infections. Plasma riche en plaquettes (PRP) est une concentration de sang autologue qui contient plus de 30 facteurs de croissance qui peuvent contribuer à l&#39;os et la cicatrisation de greffe osseuse 3-5. L&#39;application de PRP pour favoriser la régénération osseuse et de la maturation des tissus mous a été rapporté dans les cliniques de plus en plus en raison de sa forte concentration de divers facteurs de croissance libérés par les plaquettes. Plusieurs caractéristiques de PRP PRP indiquent que peuvent aussi avoir des propriétés antimicrobiennes 6-9. PRP contient un grand nombre de plaquettes, une forte concentration de leucocytes (qui peut posséder hôte de défense contre les actions des bactéries et des champignons), et plusieurs peptides anti-microbiens 7,8,10. Dans une étude récente sur une large cohorte de patients de chirurgie cardiaque, il a été révélé que l&#39;utilisation peropératoire de PRP-gel au cours de la plaie fermeture Significantly diminué l&#39;incidence de l&#39;infection par le sternum superficielle et profonde 11. Pour ces raisons et d&#39;observations, nous avons émis l&#39;hypothèse que le PRP, en plus de ses bien étudiées guérison des propriétés favorisant, possède des propriétés antimicrobiennes. Les avantages potentiels de l&#39;utilisation de PRP pour prévenir l&#39;infection peuvent inclure: (i) PRP est moins susceptible d&#39;induire une résistance par rapport à des traitements antibiotiques conventionnels. (Ii) PRP a aussi des propriétés qui favorisent la guérison qui peut avoir un effet synergique sur la prévention des infections; PRP de la guérison, la promotion de propriétés pourrait fournir un joint pour empêcher la fixation des bactéries réduisant ainsi les chances d&#39;infection par les pathogènes et les cellules humaines sont en compétition pour les surfaces de l&#39;implant 12 , 13. (Iii) PRP est intrinsèquement biocompatible et sécuritaire et exempt de risque de maladies transmissibles. Notre objectif à long terme est d&#39;utiliser PRP comme une nouvelle approche pour prévenir implant associé infections. Le but de cette étude était de préparer PRP en utilisant une approche centrifugation à deux reprises, d&#39;examiner PRP dans des propriétés antimicrobiennes in vitro, et de décrire les protocoles pour l&#39;évaluation de ces propriétés antimicrobiennes.

<table border="1">
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 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine thrombin</td>
 <td>King Pharmaceuticals, Inc </td>
 <td>60793-215-05</td>
 <td>Thrombin (bovine origin)</td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>King Pharmaceuticals, Inc</td>
 <td>60793-215-05</td>
 <td>10% calcium chloride</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>E7023</td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td></td>
 </tr>
 <tr>
 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>Tri-sodium citrate</td>
 <td>Sigma-Aldrich</td>
 <td>W302600</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryptic soy agar</td>
 <td>Fisher Scientific</td>
 <td>R01202</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Kendro Laboratory Products</td>
 <td>750043077</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe filter</td>
 <td>Millipore</td>
 <td>SLGP033RS</td>
 <td></td>
 </tr>
 </tbody>
</table>

prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

Implant associée à l&#39;infection devient de plus en plus difficile pour l&#39;industrie des soins de santé dans le monde entier en raison de la résistance croissante aux antibiotiques, la transmission de bactéries résistantes aux antibiotiques entre les animaux et les humains, et le coût élevé du traitement des infections. Dans cette étude, nous décrivons une nouvelle stratégie qui pourrait être efficace dans la prévention des infections associées aux implants basée sur les propriétés antimicrobiennes potentielles de plasma riche en plaquettes (PRP). En raison de ses propriétés bien étudiées pour favoriser la cicatrisation, PRP (un produit biologique) a été de plus en plus utilisées pour des applications cliniques, y compris les chirurgies orthopédiques, chirurgies parodontales et orale maxillo-faciale, la chirurgie, la chirurgie plastique, médecine du sport, etc PRP pourrait être une alternative de pointe pour traitements antibiotiques conventionnels en matière de prévention des infections associées implant. L&#39;utilisation de PRP peut être avantageux par rapport aux traitements antibiotiques conventionnels scnce PRP est moins susceptible d&#39;induire une résistance aux antibiotiques et PRP antimicrobienne et la guérison des propriétés favorisant peut avoir un effet synergique sur la prévention des infections. Il est bien connu que les agents pathogènes et les cellules humaines sont en compétition pour les surfaces de l&#39;implant, et les propriétés de PRP de guérison de promotion pourrait améliorer la fixation des cellules humaines réduisant ainsi les chances d&#39;infection. En outre, le PRP est intrinsèquement biocompatible et sécuritaire et exempt de risque de maladies transmissibles. Pour notre étude, nous avons sélectionné plusieurs souches bactériennes cliniques qui sont généralement trouvés dans les infections orthopédiques et a examiné si PRP a les propriétés in vitro antimicrobiens contre ces bactéries. Nous avons préparé PRP en utilisant une approche qui permet centrifugation deux fois la même concentration plaquettaire à obtenir pour tous les échantillons. Nous avons obtenu des résultats cohérents antimicrobiens et a constaté que PRP a une forte propriétés in vitro antimicrobiens contre les bactéries comme methicillin sensible et résistant à la méthicilline Staphylococcus aureus, streptocoques du groupe A, et Neisseria gonorrhoeae. Par conséquent, l&#39;utilisation de PRP peut avoir le potentiel pour prévenir l&#39;infection et à réduire le besoin de coûteux traitement post-opératoire de l&#39;implant infections nosocomiales.

Le PRP est reproductible préparés en utilisant une approche centrifugation deux fois (Figure 1). PRP est considérée comme présentant une forte (jusqu&#39;à 100 fois la réduction de UFC) dans les propriétés antimicrobiennes in vitro contre les staphylocoques résistants à la S. méthicilline (SARM) (figure 3), qui se trouve couramment dans les hôpitaux à travers le monde 14. De même, le PRP a de fortes propriétés antimicrobiennes contr...

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

Implant associée à l&#39;infection est une complication clinique importante. Cette étude décrit une approche basée sur plasma riche en plaquettes (PRP) pour prévenir les infections associées aux implants, présente le protocole pour la préparation de PRP avec la concentration de plaquettes constante, et rend compte des propriétés nouvellement identifiés antimicrobiennes de PRP et les protocoles connexes pour examiner de telles propriétés antimicrobiennes<em&gt; In vitro.</em

PRP comme une nouvelle approche pour prévenir l&#39;infection: Préparation et<em&gt; In vitro</em&gt; Propriétés antimicrobiennes de PRP

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

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JoVE Journal

Immunology and Infection

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

21.2K vues.  West Virginia University. Implant associée à l'infection est une complication clinique importante. Cette étude décrit une approche basée sur plasma riche en plaquettes (PRP) pour prévenir les infections associées aux implants, présente le protocole pour la préparation de PRP avec la concentration de plaquettes constante, et rend compte des propriétés nouvellement identifiés antimicrobiennes de PRP et les protocoles connexes pour examiner de telles propriétés antimicrobiennes

Vidéo: 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

Transduction rétrovirale des récepteurs de cellules T dans les lymphocytes T de souris

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 ...

Recherche

Immunologie et infection

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.

Présentation de contrainte de cisaillement dans l&#39;étude de l&#39;adhésion bactérienne

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 et robuste In vivo Et In vitro Démarche pour l&#39;étude de l&#39;assemblage du 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.

L&#39;utilisation de moyen d&#39;expectoration artificielle pour tester l&#39;efficacité des antibiotiques contre<em&gt; Pseudomonas aeruginosa</em&gt; Dans des conditions plus pertinents à la fibrose kystique du poumon

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.

Utilisation d&#39;<em&gt; Galleria mellonella</em&gt; Comme un organisme modèle pour l&#39;étude<em&gt; Legionella pneumophila</em&gt; Maladies infectieuses

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.

La surveillance des changements dans la membrane de polarité, Membrane intégrité, et intracellulaires d&#39;ions concentrations dans<em&gt; Streptococcus pneumoniae</em&gt; Utilisation des colorants fluorescents

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.

Analyse in vitro de la réponse immunitaire cellulaire Myd88 médiation au virus du Nil occidental souche mutante 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 ...

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.

Une approche simple et efficace de construire Mutant Vaccinia vecteurs de virus

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.

Rétrovirale transduction des cellules T auxiliaires comme une approche génétique pour étudier les mécanismes contrôlant leur différenciation et la fonction

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