The authors thank Dr. Lynda Partridge for the provision of advice and laboratory facilities.

1) Préparation de la lignée cellulaire: <ol><li> Le développement de la lignée de cellules RBL-de 2H3.1 exprimer équins α FceRI: <ol><li> En utilisant des techniques de base de culture de tissus de lignées de cellules en monocouche, transfecter des cellules RBL-2H3.1 parentales en utilisant le plasmide pEE6, portant le gène équine FcεRIα (GenBank: Y18204.1) 28. Ajouter 2 ul -1 pg de l&#39;ADN plasmidique de 0,8 ml de cellules à une densité de 1,2 x 10 7 cellules ml -1. L&#39;électroporation des cellules à 250 V 960 pF en utilisant un 0,4 cm electrocuvette puis incuber immédiatement sur la glace pendant 10 min. </li><li> Sélectionnez les cellules transformées en utilisant les médias contenant 0,4 g de sulfate de généticine G418, puis trier les cellules vivantes restantes par FACS en les étiquetant avec un anticorps fluorescent IgE. Utilisez le RBL-2H3.1 résultant lignée cellulaire exprimant FcεRIα équine de l&#39;enquête 2, 3. </li></ol></li><li> Pré-test anticorps sensibilisation: <ol><li&gt; Récolte la RBL-2H3.1 cellules exprimant FcεRIα d&#39;équidés en provenance d&#39;une boîte de Pétri de confluence. Laver ensuite remettre en suspension les cellules dans un milieu de culture à une densité cellulaire de 5x10 5 cellules ml-1. </li><li> Ajouter l&#39;IgE de l&#39;intérêt pour les cellules en suspension à une concentration finale de 1 ng ml-1, puis la plaque 100 pl de cellules sur une plaque à 96 puits à colonnes 1-6 et incuber à 37 ° C + 5% de CO 2 + 90% humidité relative pendant 16 heures. Après le temps d&#39;incubation, et avant d&#39;effectuer le test de libération, vérifier les puits sous un microscope pour bien confluence et l&#39;adhérence des cellules. </li></ol></li></ol> 2) Dosage de libération: <ol><li> Lavage des cellules: <ol><li> Un tampon de sortie chaud (25 mM de PIPES, du chlorure de sodium 120 mM, 5 mM de chlorure de potassium, 0,04 mM de chlorure de magnésium et 1 mM de chlorure de calcium) à 37 ° C pour permettre le lavage des cellules en douceur. </li><li> Laver les cellules en tapotant la plaque pour enlever médias cellulaires et ajouter 100 ul chaud, 37 ° C, tampon de sortie. Répéter deux fois. </li></ol></li><li> Défi antigène: <ol><li> Préparer une série de dilutions de l&#39;antigène (NIP-HSA ou DNP-HSA) de 0 ng ml -1, 0,1 ng ml -1, 1 ng ml -1, 10 ng ml -1, 100 ng ml -1, 1 000 ng ml -1, 10 000 ng ml -1 dans le tampon de sortie et chaud à 37 ° C. </li><li> Après le second lavage de cellules, éliminer les médias et remplacé par 100 ul de la solution d&#39;antigène. Veiller à ce que les puits dans la même rangée (A1-6, par exemple) ont la même concentration ajoutée à leur antigène. </li><li> Mettre en place un contrôle négatif à la ligne A en ajoutant 0 antigène -1 ml ng. Ajouter augmentation de la concentration de l&#39;antigène dans les rangées (BG), suivie par triton x-tampon (5% de Triton X-100) dans les cellules de rangées H à lyser les cellules pour être utilisé en tant que contrôle positif. Incuber à 37 ° C pendant 20 min pour permettre aux cellules de libérer ses médiateurs. </li></ol></li><li> Configuration du contrôle de puits individuels: <ol><li> Après l&#39;incubation, transférer 50 pi de surnageant de cellules à l&#39;autre moitié de la plaque (A1-6 puits à puits A7-12, etc.). Jeter les 50 ul de surnageant restant et le remplacer par 50 ul de Triton X-tampon pour permettre la mesure de la quantité de médiateurs libérés à chaque puits dans les colonnes 7 à 12 en pourcentage du total des médiateurs à l&#39;intérieur des cellules dans la colonne 1-6 . </li></ol></li><li> Substrat enzymatique: </li><li> Ajouter 50 ul de substrat de β-hexosaminidase (mM 4-nitrophényl N-acétyl-β-D-de glucosaminide préparé dans du DMSO dilué jusqu&#39;à 2 mM en l&#39;ajoutant à un tampon de citrate 0,2 M d&#39;acide citrique et 0,2 M d&#39;acétate de sodium, pH 4,5 à 50) dans tous les puits de la facilité de conversion du substrat de 4-nitrophénol par l&#39;enzyme β-hexosaminidase. Incuber les plaques à 37 ° C pendant 2 heures. </li></ol><img alt="Figure 2.4.1" fo:content-width="4in" src="/files/ftp_upload/52222/52222fig241.jpg" width = &quot;400&quot; /&gt; <ol start="5"><li> Mettre fin à la réaction: <ol><li> Arrêter la réaction par l&#39;ajout de 150 ul de tampon Tris (1 M de Tris-HCl, pH 9) à chaque puits comme le pH élevé de la mémoire tampon arrête la réaction et le transforme en 4-nitrophénol de couleur jaune. </li></ol></li><li> La lecture et l&#39;analyse des résultats: <ol><li> Lire la plaque à l&#39;aide d&#39;un spectrophotomètre de plaque à 405 nm pour mesurer l&#39;absorbance de la couleur jaune. Calculé le pourcentage de β-hexosaminidase libéré en utilisant la formule suivante: </li></ol></li></ol><img alt="Figure 2.6.1" fo:content-width="1in" src="/files/ftp_upload/52222/52222fig261.jpg" width="100" /><ol start="2"><li> Appliquer cette formule à chaque puits, après quoi une moyenne est calculée pour chaque ligne. A1 et A7 représentent la position des puits dans la plaque à 96 puits. Tracer la courbe de pourcentage de β-hexosaminidase de presse (qui correspond à la libération totale du médiateur) contantigène NST de concentration 2, 3. </li></ol>

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The authors declare that they have no competing financial interests in this paper.

En résumé, les résultats de cette enquête ont montré que lorsque les cellules RBL-2H3.1 exprimant FcεRIα équins sont sensibilisées avec équine IgE et remis en cause par un antigène, ils donnent un pic de libération de médiateur de 36,68% ± 4,88% du montant total de médiateur à l&#39;intérieur du cellules, par rapport à la RBL-2H3.1 cellules parentales non exprimant équine FcεRIα. Ainsi, ce test fournit un outil utile pour étudier et étudier les réponses allergiques é...

Allergie est connue depuis des millénaires. Un traitement de l&#39;asthme a été décrit dans le texte médical égyptien antique connu sous le papyrus Ebers (~ 1550 avant notre ère) et discuté remèdes pour traiter 7. allergie est aujourd&#39;hui considéré comme une réponse d&#39;hypersensibilité de type I, où le type T cellulaire auxiliaire 2 (TH 2) du bras du système immunitaire dirige la production d&#39;immunoglobulines E (IgE), des anticorps en réponse à des antigènes environnementaux appelés allergènes. Ce sont diverses substances qui interagissent couramment avec les cellules du système immunitaire et stimuler la synthèse et la sécrétion de cytokines pro-inflammatoires, y compris l&#39;interleukine-4 et l&#39;interleukine-13, 8, 9 comme ne particules dans les particules de fumée de cigarette ou diesel échappement qui augmente la synthèse des IgE 10. La hausse des manifestations allergiques dans les pays industrialisés au cours des 50 dernières années a été attribué à une combinaison de l&#39;effet depolluants de l&#39;environnement et une tendance à un environnement plus aseptisé, qui se combinent pour décaler la réponse immunitaire vers un profil prédominé par Th 2 cytokines, telle que proposée par la &#39;hypothèse hygiénique »11. Comme mentionné ci-dessus, l&#39;homme ne sont pas les seuls mammifères affligés par une allergie. Notamment les chevaux et les chiens peuvent aussi développer des réactions allergiques classiques et une étude de 12 a montré que, comme chez les humains, l&#39;allergie équin est attribuée à des facteurs génétiques et environnementaux. En conséquence, ces animaux présentent de bons modèles pour étudier l&#39;interaction entre les facteurs génétiques et environnementaux de l&#39;allergie, la progression de la sensibilisation à la maladie et les stratégies d&#39;intervention possibles une fois les manifestations cliniques sont fixés dans En 1887, Stömmer était la première personne pour décrire la similitude entre l&#39;homme et l&#39;asthme équine 13, l&#39;effet de l&#39;histamine sur le système cardio-vasculaire équine esttrès similaire à celui des humains 14. Les chevaux sont aussi la pierre angulaire de l&#39;industrie des courses de chevaux, qui vaut 72 milliards de dollars, un chiffre d&#39;affaires de paris de 115 milliards de dollars par an 15. La plupart des chevaux de course contemporains sont des descendants du petit nombre de chevaux arabes élevés par Lady Anne Blunt à partir de 1878. Chevaux de course modernes sont souvent consanguins à sélectionner pour les capacités de performance. Ils sont sujets à des troubles génétiques, dont l&#39;un est leur susceptibilité à monter des réponses allergiques. Ils ont aussi 1000 fois plus élevés que les niveaux d&#39;IgE sériques même les êtres humains plus sévèrement allergique 16. réactions allergiques à cheval sont se manifeste habituellement par une hypersensibilité insecte morsure (IBH) 17, 18. IBH résultats dans des dermatoses dues aux piqûres d&#39;insectes forment dans le genre Culicoides. Une autre forme de la maladie allergique équine est obstructions des voies respiratoires récurrentes (RAO), cela se manifeste dans les poumons et les voies respiratoires. Elle est caractérisée par une respiration sifflante et laboratoirerespiration ORed. RAO se produit généralement en réponse à spores de moisissures, et des niveaux élevés d&#39;IgE spécifiques des allergènes ont été enregistrés chez les chevaux atteints de RAO dans une étude 19 mais une autre enquête n&#39;a pas confirmé cette 20. Des études sur l&#39;allergie équine ont tourné autour de l&#39;essai de contrôle et de neutraliser équine IgE par le développement d&#39;anticorps anti-équin IgE monoclonal (mAb) 21, 22. En outre, l&#39;étude de 23 traite de la production de domaines extracellulaires de l&#39;α de l&#39;équin haute affinité Fc récepteur chaîne (FcεRIα) récepteur dans le but de détecter et de quantifier le sérum équin IgE. Une étude connexe par Ledin 24 traite d&#39;une nouvelle approche visant à neutraliser les IgE sériques en amorçant le système immunitaire en utilisant un auto / non-soi immunogène. Toutes ces études, cependant, manquait un dosage efficace pour tester l&#39;innocuité et l&#39;efficacité de leurs protocoles. Dans cet article, nous présentons un tel sys d&#39;essaisystème applicable à l&#39;étude des stratégies diagnostiques et thérapeutiques pertinentes pour le système équin, où β-hexosaminidase libération, comme un indicateur de médiateur de la dégranulation cellulaire, a été évaluée sur des cellules RBL-2H3.1 exprimant FcεRIα équine. Ce protocole est basé sur des publications antérieures 25, 4, 5, 2, 3, décrivant l&#39;ingénierie de cellules RBL transfectées avec le gène codant le domaine de liaison du récepteur IgE de haute affinité pour les IgE provenant d&#39;espèces différentes. Le protocole explique comment effectuer un test de libération de β-hexosaminidase, dont les résultats sont présentés en moyenne ± écart-type d&#39;expériences en triple. Le dosage de la libération a été développé par Siraganian et le crochet 25 pour étudier allergie humaine. Le groupe de laboratoire dirigé par le Dr Reuben Siraganian également développé la lignée cellulaire RBL. Ces cellules RBL ont été développés pour exprimer la FcεRIα humain et le protocole a été publié par 4. La dernière piècedu dosage fournie avec le développement du plasmide pSV dans le papier par Neuberger 26 qui décrit la production d&#39;un anticorps IgE par clonage de son gène de chaîne lourde en aval d&#39;un gène de la souris pour une région variable d&#39;IgE qui cible l&#39;haptène 4-hydroxy-3 nitro-phénacétyle (NP), l&#39;anticorps chimérique résultant était pleinement fonctionnel. La possibilité de développer tout IgE ciblant le même haptène, tout en clonant son récepteur sur la surface des cellules RBL abouti à la normalisation du dosage qui en fait un protocole utile pour mesurer la dégranulation des basophiles. Le test a des avantages et des inconvénients. Les avantages du dosage est son adaptabilité à être utilisé dans un système de mammifère, notre laboratoire a ainsi utilisé pour tester la dégranulation dans les systèmes humains, canins, équins, ce qui est réalisable simplement par synthèse d&#39;IgE de l&#39;organisme et le clonage de son récepteur sur la surface des cellules RBL. D&#39;autre part,les inconvénients de l&#39;essai est que les cellules RBL sont très sensibles aux variations thermiques, mécaniques et PH, ce qui leur donne une variation des niveaux de dégranulation au sein de la même dosage. Il est donc fortement conseillé que les essais sont toujours répétées en triple, puis en prenant la moyenne de leur part. En outre, les cellules RBL ont tendance à se déplacer vers un phénotype non-libération si elles sont laissées en culture de tissu pendant des périodes prolongées (&gt; 10 semaines) 27, ce qui rend leur entretien lourd. Elles sont également sujettes à des infections par des bactéries de mycoplasmes, qui ne sont pas visibles à partir d&#39;un microscope optique et ne modifient pas la morphologie de la cellule, mais cela changerait radicalement leurs niveaux de dégranulation. Ainsi des tests réguliers de mycoplasmes sont nécessaires.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>RBL-2H3.1 Expressing Equine Fc&#949;RI&#945;</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>Equine IgE anti NIP-HSA</td>
			<td>-</td>
			<td>-</td>
			<td>Produced in the lab</td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Sigma</td>
			<td>CLS3595</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Multi Channel Pipette</td>
			<td>Anachem</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Galaxy R</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4Hydroxy-5-iodo-3-nitrophenylacetic acid</td>
			<td>Cambridge Research Biochemicals</td>
			<td>N-1070-1</td>
			<td>NIP-OH was conjugated with Human Serum Albumin to make NIP-HSA in the lab</td>
		</tr>
		<tr>
			<td>Dinitrophenyl Conjugated to Human Serum Albumin</td>
			<td>Sigma</td>
			<td>A6661</td>
			<td>Abbreviated DNP-HSA</td>
		</tr>
		<tr>
			<td>Plate Spectrophotometer</td>
			<td>Anthos Labtec HT2</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>Sigma</td>
			<td>P1851</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Triton x100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>-</td>
		</tr>
		<tr>
			<td>4-nitrophenyl N-acetyl-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>Stock solution called &#946;-hexosaminidase substrate was 50mM prepared in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma</td>
			<td>251275</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma</td>
			<td>S7670</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

development of an in vitro model system for studying the interaction of equus caballus ige with its high-affinity receptor fc&#949;ri

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

Les cellules RBL-2H3.1 parentales et celles transfectées avec le gène du récepteur de FcεRIα équine ont d&#39;abord été sensibilisées avec des IgE de souris anti- DNP-HSA et la provocation avec l&#39;antigène DNP-HSA. IgE de souris se lie au récepteur de rat endogène dans les deux lignées cellulaires et agit comme un contrôle pour tester la viabilité de la libération de deux lignées cellulaires pour libérer des médiateurs (figure 1 A) ainsi. Ceci est un test important et doit être ef...

Watch this Scientific Journal Video about Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÎµRI at JoVE.com

Development of an &lt;em&gt;in vitro&lt;/em&gt; model system for studying the interaction of &lt;em&gt;Equus&lt;/em&gt; &lt;em&gt;caballus&lt;/em&gt; IgE with its high-affinity receptor Fc&amp;#949;RI

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Développement d&#39;un<em&gt; In vitro</em&gt; Système modèle pour l&#39;étude de l&#39;interaction de<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE avec son récepteur de haute affinité FcsRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

development-an-vitro-model-system-for-studying-interaction-equus

Research

JoVE Journal

Immunology and Infection

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

12.3K vues.  King Abdulaziz University. The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine FcεRIα gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Vidéo: Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Développement d&#39;un marqueur sélectionnable négatif pour les Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Recherche

Immunologie et infection

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

Dans la formation de biofilms in vitro dans une diapositive Chambre de 8 puits

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Mise en place d&#39;un<em&gt; In vitro</em&gt; Système d&#39;étudier le comportement intracellulaire de<em&gt; Candida glabrata</em&gt; En droits macrophages THP-1

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

A Novel<em&gt; In vitro</em&gt; Modèle pour l&#39;étude des interactions entre les droits du sang total et de l&#39;endothélium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

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 ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Un<em&gt; In Vitro</em&gt; Modèle de mesure de réponses immunitaires contre le paludisme dans le contexte du VIH co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Etudier communautés microbiennes<em&gt; In Vivo</em&gt;: Un modèle d&#39;interaction hôte intermédiaire Entre<em&gt; Candida albicans</em&gt; Et<em&gt; Pseudomonas aeruginosa</em&gt; Dans les Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Un<em&gt; In vitro</em&gt; Modèle de la barrière hémato-encéphalique utilisant la spectroscopie d&#39;impédance: un regard sur T Cell-endothélial cellulaire Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualisation des effets des expectorations sur le développement du biofilm Utilisation d&#39;un modèle Chambered lamelle couvre-objet

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Développement d&#39;un ADN Système économique Livraison par « Acufection » et son application à la recherche de la peau

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...