Ce travail a été soutenu par le Transregio Sonderforschungsbereich 19 (SFB/TR19, C2) de la Deutsche Forschungsgemeinschaft (DFG), le ministère fédéral de l&#39;Économie et de la Technologie du projet ZIM-KF 2727801MD0 et le Centre de Compétence Innovation - réponses immunitaires humorales dans les maladies cardio-vasculaires ( ZIK-RANDONNÉE, BMBF FKZ 03Z2CN12) du Ministère fédéral de l&#39;Education et de la Recherche, Allemagne. Logement et des expériences avec des animaux ont été effectuées en conformité avec les recommandations de la Société pour la science des animaux de laboratoire (Gesellschaft für Versuchstierkunde, GV-SOLAS) et la Fédération européenne des animaux de laboratoire Science Association (FELASA).

1. Isolation IgG des échantillons de patients <ol><li> Préparer des mini-colonnes immunoadsorption en remplissant 3 ml anti-IgG sépharose par 2 ml de plasma EDTA du patient dans une colonne vide Econo Pac. IgG isolement nécessite au moins 2 ml de plasma du patient. </li><li> Placer un filtre sur le dessus de la sépharose anti-IgG, couper l&#39;extrémité inférieure de la colonne et appuyez sur le filtre pour atteindre un débit d&#39;environ 1 goutte / sec. Laissez la solution de conservation à court de la colonne. </li><li> Laver la colonne avec 3 volumes de NaCl 0,9% par volume de plasma. </li><li> Pipeter le plasma sur la colonne. Lorsque le plasma est complètement immergé dans la sepharose anti-IgG, laver avec le même volume de NaCl 0,9%. </li><li> Volume de pipette plasma une de HCl 0,2 M glycine, pH 2,8 sur la colonne et de laisser tremper dans la sépharose. Ajouter un autre volume de la glycine HCl sur la colonne. Recueillir le débit à travers un tube de 15 ml et ajuster le pH à 7,3 avec 0,5 M de Tris-HCl, pH 8,0. </li><li> Pour régénérer la colonne, Laver avec des volumes de plasma HCl 3 glycine. Après un dernier lavage avec deux volumes de NaCl à 0,9%, la colonne peut être utilisée pour l&#39;échantillon de plasma suivant. En variante, la colonne peut être rempli avec une solution de préservation et conservé à 4 à 8 ° C pendant un maximum de 4 semaines. </li><li> Destiné à être utilisé sur des cardiomyocytes, la fraction IgG collectés doit être dialysée contre du tampon expérimentale (EB). Pour la préparation de EB, ajouter 117 mM de NaCl, 2,8 mM KCl, 0,6 mM de MgCl2, 1,2 mM KH 2 PO 4, 1,2 mM de CaCl2, 10 mM d&#39;HEPES et 20 mM de glucose à 1 L d&#39;eau distillée sur un agitateur magnétique et ajuster le pH à 7,3. </li><li> Remplir la fraction IgG à un tube d&#39;ester de cellulose membrane de dialyse ayant un poids moléculaire de couper 100 kDa. Placer le tube dans 1 L EB et remuer lentement avec un agitateur magnétique à 4 ° C. Après 8 heures, transférer le tube de dialyse dans 3 L d&#39;EB frais et dialyse pour une autre h 20 à 4 ° C. </li><li> À la suite de dialyse, de la chaleur de l&#39;échantillon pendant 30 min à 57 ° C dans un bain d&#39;eau à inactivatfacteurs du complément e. Déterminer la concentration totale en IgG et préparer des aliquotes contenant 330 ug de chacun des IgG. Lors de l&#39;ajout de l&#39;échantillon à cardiomyocytes de mesure (étape 4.3), remplir les aliquotes de 1 ml avec EB. Aliquotes non diluées peuvent être conservés à -20 ° C jusqu&#39;à utilisation ultérieure. </li></ol> 2. Isolement de cardiomyocytes de rats <ol><li> Pour la préparation de Ca 2 +-tampon d&#39;isolement libre (IB), ajouter 110 mM de NaCl, 2,6 mM KCl, 1,2 mM MgSO 4, 1,2 mM KH 2 PO 4, HEPES 20 mM et 11 mM de glucose à 1 L d&#39;eau distillée en utilisant un champ magnétique agitateur. Ajuster le pH à 7,4 à la température ambiante et filtrer à travers un filtre de 0,2 um bouteille supérieure pour la stérilisation. </li><li> Remplir 80 ml d&#39;IB dans le réservoir supérieur d&#39;un système de perfusion de Langendorff thermostaté. Ajuster les réglages du thermostat pour maintenir une température tampon de 37 ° C et aérer tampon avec 95% 2 O / 5% de CO 2 pendant 30 min. </li><li> Peser environ 10.000 - 12.000 U de collagenase de type II, 175 mg d&#39;acides gras libres BSA et dissoudre dans 20 ml contenant IB 22,5 pi d&#39;une solution 100 mM CaCl stock 2. Contrôler le pH du tampon d&#39;isolement dans le réservoir supérieur et l&#39;ajuster si nécessaire. </li><li> Anesthésier un rat Wistar de 175 - 200 g à 375 mg / kg poids corporel thiopental. Ajouter 2500 UI d&#39;héparine à la solution de thiopental. Après thoracotomie, au cœur d&#39;accise soigneusement avec une crosse de l&#39;aorte intacte et le transférer dans glacée NaCl à 0,9%. Nettoyez le cœur du sang et des tissus environnants et le transfert de frais de NaCl 0,9%. Exposer l&#39;aorte, ouvrez le réducteur de débit du système de Langendorff pour obtenir une vitesse de chute des 2-3/sec et fixez le coeur de la canule. </li><li> Perfuser le coeur jusqu&#39;à 3 min avec un débit de 1 goutte / sec pour laver les résidus de sang. Commenceront à circuler le BI dans le système de Langendorff. Retirer 20 ml de tampon du réservoir supérieur, remplissez la solution de collagénase et de remplissage de tampon autant que nécessaire pour atteindre 80 ml. Faire circuler le c<html> solution pour un autre ollagenase min 27. Débit doit être contrôlé périodiquement et maintenue à 1 goutte / sec en utilisant le réducteur de débit. </li><li> Détachez le coeur de la canule, propre à partir de l&#39;aorte et les oreillettes et les couper en petits morceaux. Laisser la solution de collagénase à courir dans le réservoir inférieur, réduisez le volume à un maximum de 40 ml et encore digérer les ventricules hachées pendant 10 à 15 min sous aération continue avec 95% O 2/5% de CO 2. Ensuite, filtrer la suspension de cellules à travers une gaze 200 um maille dans un tube de 50 ml. </li><li> Restaurer le Ca 2 + contenu des cellules en 3 étapes: centrifuger la suspension cellulaire à 43 xg pendant 2 min à température ambiante et remettre les cellules en IB de 10 ml contenant 200 uM CaCl 2. Centrifuger de nouveau et remettre en suspension les cellules dans 10 ml IB avec 500 uM CaCl 2. Après une centrifugation finale remettre en suspension dans les cardiomyocytes EB filtrée stérile (voir 1.8) à une densité de 50.000 cellules / ml. </li></ol>e_title &quot;&gt; 3. Fura-2 Coloration des cardiomyocytes <ol><li> Enduire une lamelle 4 bien chambré avec 10 ug par la laminine bien et attendez qu&#39;il soit complètement sec. </li><li> Remplir 1 ml de la suspension des cardiomyocytes dans chaque puits à l&#39;aide d&#39;une micropipette avec une pointe de coupe. Laisser les cellules adhérer pendant 1 h à température ambiante. </li><li> Diluer 10 ul de la 1 mg / ml Fura-2 AM solution mère dans 5 ml EB. Conserver la solution de coloration dans l&#39;obscurité. </li><li> Enlever cellules tampons et les personnes seules de la lamelle et la pipette 0,5 ml de la solution de coloration dans chaque puits. Incuber les cellules pendant 10 min sous agitation modérée. Par la suite, enlever la solution de coloration, laver les cellules une fois avec 1 ml EB et enfin remplir EB 0,5 ml dans chaque puits. Evitez la lumière directe pendant le processus de coloration et de toujours garder les cellules colorées dans l&#39;obscurité. </li></ol> 4. Enregistrement de raccourcissement de la cellule et Ca 2 + transitoires <ol><li> Placez la lamelle chambré sur une m fluorescence inverseicroscope relié à un myocyte calcium et d&#39;enregistrement de la contractilité. Positionner une électrode de mesure avec un tube d&#39;afflux et écoulement dans le premier puits. Cardiomyocytes Superfuse avec 1 ml / min et EB commencer la stimulation électrique (20 V, 1 Hz, 5 msec). Permettre aux cellules de s&#39;adapter à la stimulation pendant environ 2 min. </li><li> Choisissez un des cardiomyocytes tige intacte avec des stries en forme claire et constante contraction. Positionner la cellule dans le centre du champ visuel et ouvrir le canal de caméra vidéo. Orientez la cellule horizontalement dans la zone vidéo en tournant l&#39;adaptateur cadrage cellule et déplacer la platine du microscope. </li><li> Ajuster la détection de bord des éléments de commande de la zone vidéo (figure 2), ouvrir le canal de fluorescence et d&#39;enregistrement de 10 à 15 pour obtenir des contractions du raccourcissement de la cellule initiale et transitoire du Ca 2 +. </li><li> Fermer le canal de lumière microscope pour éviter le blanchiment de la tache de fluorescence. Mettez le débit à travers de EB à l&#39;échantilloncontourner avec une vanne 3 voies et les cardiomyocytes superfuse avec 1 ml d&#39;IgG des patients. Arrêter la pompe juste avant que l&#39;air atteint le puits. </li><li> Ouvrez le canal de lumière et enregistrer un autre 10 à 15 contractions pour obtenir la réponse cellulaire aiguë. Interrompre l&#39;enregistrement et fermer le canal de lumière à nouveau. Répétez l&#39;enregistrement après 2 et 5 min. </li><li> Retirez l&#39;électrode du puits. Nettoyez les tubes à fond avec EB et continuer avec le bien suivant de la lamelle chambré. </li><li> Analyser le raccourcissement de la cellule et le Ca 2 + avec le logiciel transitoire IonWizard utilisant la &quot;Edge-Length/Length&quot; et le &quot;fura 2-numérique soustraite / Ratio&quot; fenêtre, respectivement. Calculer la variation pour cent du raccourcissement de la cellule initiale et transitoire du Ca 2 + de la hauteur de crête visée à la ligne de base (bl% de pic h) pour l&#39;aigu, le 2 min et la réponse 5 min. </li></ol>

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La méthode présentée offre un moyen approprié pour détecter fonctionnellement efficaces autoanticorps cardiaques chez les patients atteints de DCM d&#39;origine incertaine. En comparaison à d&#39;autres méthodes, par exemple la détection d&#39;anticorps fonctionnels actifs contre la β1-adrénergiques par leur impact sur ​​les taux d&#39;AMPc 6, notre méthode est indépendante d&#39;un épitope spécifique. Bien sûr, il existe d&#39;autres épitopes essais indépendants décrits dans la...

<table border="1"><tbody><tr><td> Nom du réactif / équipement </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Immunosorba préservation solution </td><td> Fresenius Medical Care </td><td> 903609211 </td><td></td></tr><tr><td> Collagénase de type 2 </td><td> Cell System </td><td> LS004176 </td><td></td></tr><tr><td> BSA, acides gras libres </td><td> Sigma-Aldrich </td><td> A6003 </td><td></td></tr><tr><td> La laminine </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Fura-2 AM </td><td> Sigma-Aldrich </td><td> F0888 </td><td> 1 mg / ml dans du DMSO </td></tr><tr><td> Econo Pac Colonnes de chromatographie </td><td> BioRad </td><td> 732-1010 </td><td></td></tr><tr><td> Spectra / Por Biotech Ester de cellulose de dialyse Membrane </td><td> Spectrumlabs </td><td> 131417 </td><td></td></tr><tr><td> 4 bien chambré lamelle couvre-objet </td><td> Nunc </td><td> 155383 </td><td></td></tr><tr><td> Le calcium des myocytes et système d&#39;enregistrement contractilité </td><td> IonOptix </td><td></td><td></td></tr><tr><td> Logiciel d&#39;analyse IonWizard 6,0 </td><td> IonOptix </td><td></td><td></td></tr></tbody></table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

La cardiomyopathie dilatée (DCM) est l&#39;une des principales causes de l&#39;insuffisance cardiaque chez les jeunes adultes 1. Bien que la prédisposition génétique et l&#39;exposition à des substances toxiques sont des causes connues de cette maladie chez environ un tiers des patients, l&#39;origine de DCM reste largement incertain. Dans un nombre important de ces patients, des auto-anticorps dirigés contre les épitopes cardiaques ont été détectés et sont soupçonnés de jouer un rôle central dans l&#39;apparition et la progression de la maladie 2,3. L&#39;importance de l&#39;auto-anticorps cardiaques est soulignée par une amélioration hémodynamique chez les patients DCM après élimination des auto-anticorps par immuno 3-5. Une variété d&#39;antigènes spécifiques ont déjà été identifiés et 2,3 anticorps contre ces cibles peuvent être détectées par des dosages immunologiques. Toutefois, ces tests ne peuvent pas distinguer entre la stimulation (et donc fonctionnellement efficace) et le blocage auto-anticorps. Il est en augmentation evidenCE que cette distinction est cruciale 6,7. Il peut également supposer que les objectifs fixés pour un certain nombre d&#39;anticorps cardiotropes sont encore non identifiés et ne peuvent donc pas être détectée par des dosages immunologiques. Par conséquent, nous avons établi une méthode pour la détection des anticorps fonctionnellement actifs cardiotropes, indépendamment de leur antigène respectif. Le contexte de la méthode est la forte homologie habituellement observée pour les régions fonctionnelles des protéines cardiaques chez les mammifères entre 8,9. Ceci suggère que les anticorps dirigés contre les antigènes cardiaques humaines réaction croisée avec les cellules cibles non humaines, ce qui permet des tests d&#39;IgG de patients DCM sur les cardiomyocytes de rats adultes. Notre méthode se compose de 3 étapes: d&#39;abord, l&#39;IgG est isolée du plasma du patient à l&#39;aide de sépharose couplées à des anticorps anti-IgG obtenues à partir des colonnes immunoadsorption (PlasmaSelect, Teterow, Allemagne). Deuxièmement, les cardiomyocytes adultes sont isolés par perfusion de collagénase dans un appareil de perfusion de Langendorff en utilisant aprotocole modifié à partir de travaux antérieurs 10,11. Les cardiomyocytes obtenus sont joints à la laminine revêtues de couvre-objets cloisonnées et colorées avec du Fura-2, un colorant fluorescent sélectif de calcium qui peut être facilement introduit dans la cellule d&#39;observer calcium intracellulaire (Ca 2 +) contenu 12. Dans la dernière étape, l&#39;effet du patient IgG sur le raccourcissement de la cellule et Ca 2 + transitoires de cardiomyocytes le terrain ont stimulé est suivi en ligne en utilisant un calcium du commerce des myocytes et système de surveillance de la contractilité (IonOptix, Milton, MA, USA) connecté à un fluorescent standard inverse microscope.

Deux exemples pour la mesure de inotropie cellulaire dans le champ stimulé cardiomyocytes adultes (figure 2) en utilisant un myocyte Ca 2 + et d&#39;enregistrement de la contractilité sont donnés ci-dessous. Figure 3 donne une impression d&#39;une mesure de contrôle, tandis que la figure 4 l&#39;effet des anticorps cardiodépresseur d&#39;un patient avec du DCM est représenté. Dans les deux exemples, le shortening cellule in...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

La méthode présentée offre un moyen de détecter les anticorps fonctionnels efficaces cardiotropes dans le plasma des patients atteints de cardiomyopathie dilatée, indépendamment de l&#39;antigène spécifique, en analysant l&#39;impact de l&#39;immunoglobuline patient isolé sur raccourcissement cellulaire et transitoires de calcium intracellulaire dans les cardiomyocytes isolés de rats.

Mesure des effets sur la fonction cellulaire anticorps de cardiomyocytes isolés

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

measurement-antibody-effects-on-cellular-function-isolated

Research

JoVE Journal

Immunology and Infection

11.8K vues.  University Medicine Greifswald. La méthode présentée offre un moyen de détecter les anticorps fonctionnels efficaces cardiotropes dans le plasma des patients atteints de cardiomyopathie dilatée, indépendamment de l'antigène spécifique, en analysant l'impact de l'immunoglobuline patient isolé sur raccourcissement cellulaire et transitoires de calcium intracellulaire dans les cardiomyocytes isolés de rats.

Vidéo: Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Mesure de cytosolique de Ca 2 + En isolé lymphatique contractiles

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Recherche

Immunologie et infection

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Un essai de microplaques pour évaluer les effets des produits chimiques sur RBL-2H3 mât dégranulation: Effets de Triclosan sans utilisation d&#39;un solvant organique

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Les méthodes de détection quantitative d&#39;anticorps induite par l&#39;activation du complément sur les globules rouges

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Analyser les effets des cellules stromales sur le recrutement des leucocytes de débit

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Cytométrie en flux Analyse de la particule liée Bet v 1 Allergène en PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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 ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

L&#39;identification d&#39;un épitope linéaire des cellules B monoclonales d&#39;anticorps reconnus par le peptide à balayage assistée

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Test de tolérance au Glucose intrapéritonéal, mesure de la fonction pulmonaire et la Fixation du poumon pour étudier l’Impact de l’obésité et des troubles du métabolisme sur résultats pulmonaires

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Évaluation des effets de médicaments à base d’anticorps murins de la moelle osseuse et d’Activation des macrophages péritonéaux

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Une méthode pour la mesure de la fonction des glandes salivaires chez les souris

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...