This research was supported by a grant from the NSERC Discovery program grant number RGPIN 418522-2013. We thank R. Kilgour for helpful discussions, and Katelin Gresty and Dr. A. Berghdal for providing the mouse muscle sections.

Recherche avec des échantillons de sang humain a été approuvé par le Conseil d&#39;examen éthique de l&#39;Université Concordia, le numéro de certificat 10000618. Le travail sur les muscles de la souris a été approuvé par le Conseil d&#39;examen éthique de l&#39;Université Concordia, numéro de certificat 2010BERG. 1. Isolement PBMC à partir de sang REMARQUE: Effectuer cette procédure dans une enceinte de biosécurité utilisant une technique stérile et de l&#39;équipement du fabricant stérilisé. <ol><li> Verser soigneusement 10 à 15 ml de sang complet à partir des tubes héparinés (anti-coagulant) collecte de sang dans un tube conique de 50 ml stérile. Pour les déversements de sang mineurs, essuyer avec ddH 2 O et 70% EtOH en utilisant des tissus de nettoyage. </li><li> On dilue le sang dans le tube conique à un rapport 1: 1 avec le tampon phosphate salin (PBS 1X) pH 7,4. Assurez-vous que le volume total maximum ne dépasse pas 30 ml. </li><li> Mélanger doucement à l&#39;aide d&#39;une pipette sérologique pistolet en évitant les bulles. NOTE: Ne pas mélanger en inversant le tube pour éviter un sangccumulation dans le bouchon et l&#39;infiltration subséquente pendant la centrifugation. </li><li> Ajouter 13 ml de Ficoll-Paque (voir Matériels et table Équipement), à un nouveau tube conique de 50 ml de capacité. Gardez le tube vertical dans le rack. </li><li> Aide d&#39;une pipette de transfert, une prise de sang dilué, toucher la pointe de pipette de transfert à la paroi intérieure du tube près du sommet. Avec une pression lente et régulière, transférer le sang le long de la paroi intérieure du tube formant une couche de sang au-dessus de la couche de saccharose. Effectuez cette opération plusieurs fois jusqu&#39;à ce que tout le sang a été transféré. </li><li> Boucher le tube hermétiquement et centrifuger le tube à la température ambiante pendant 30 min à 700 xg dans un rotor swing avec l&#39;ensemble de l&#39;accélération moyenne à 5, et la décélération mis à zéro. </li><li> Sortez lentement le tube et sans déranger les couches, le ramener à l&#39;enceinte de sécurité biologique. </li><li> Recueillir soigneusement la couche leucocytaire (mince couche blanche trouble), où se trouvent les CMSP, placé entre le PBS / plasma une couches de Ficoll en utilisant une pipette de transfert. Éviter de recueillir couche de saccharose et de ne pas déranger la couche de globules rouges. </li><li> Transférer la couche leucocytaire dans un nouveau tube de 50 ml conique stérile. Il peut prendre plusieurs fois de répéter l&#39;étape 1.8 de recueillir toutes les CMSP. </li><li> Ajouter PBS pH 7,4 au tube avec le CMSP et remplir jusqu&#39;à la marque 45 ml. Bien agiter tube. Ne pas vortex. </li><li> Lavez 1: Centrifugeuse pendant 15 min à 480 xg à la fois avec une accélération maximale et la décélération réglé à 9. Observez formation d&#39;un culot de PBMC au fond du tube conique. </li><li> Rejeter le PBS (surnageant) dans un bécher en plastique avec 10 ml d&#39;eau de Javel. </li><li> Desserrer culot cellulaire par doucement &quot;rayonnage&quot; contre une surface ondulée (c.-à-rack vide). Ne pas vortex. </li><li> Ajouter 25 ml de PBS frais pH 7,4 au tube. Si plus d&#39;un tube est en cours, en commun les pellets ensemble dans cette étape. </li><li> Lavez 2: Centrifugeuse le tube pendant 12 min à 480 xg with la fois l&#39;accélération et la décélération maximale fixé à 9. </li><li> Rejeter le PBS (surnageant) dans le bécher en plastique avec un peu d&#39;eau de Javel. </li><li> Doucement &quot;rack&quot; contre une surface ondulée (c.-à-rack vide). Ne pas vortex. </li><li> Ajouter 25 ml de PBS frais dans le tube. <ol><li> Sortez 50 pi des cellules et transférer dans un tube à centrifuger pour le nombre de viabilité. </li><li> Ajouter une quantité égale (50 pi) de bleu trypan (une tache de viabilité) et pipette de haut en bas pour mélanger doucement. </li></ol></li><li> Sortez 10 pi et le transférer sur un hémocytomètre pour vérifier la viabilité des cellules par ml. Notez le nombre de cellules / ml. </li><li> Sortez la quantité désirée de cellules et centrifuger le tube pendant 12 min à 480 xg à la fois avec une accélération maximale et la décélération réglé sur neuf. </li><li> Rejeter le PBS (surnageant) dans le bécher avec l&#39;eau de Javel. </li><li> Doucement &quot;rack&quot; contre une surface ondulée (c.-à-rack vide). Ajouter 80 pi de PBS dans le tube. </li></ol> 2. Faire le diaporama PBMC <ol><li> Placer 80 ul des cellules sur une lame de microscope. Enduire la goutte à l&#39;aide d&#39;une autre lame ou placer deux gouttes de 40 ul de chacune des deux extrémités de la glissière. </li><li> Laissez diapositive dans l&#39;enceinte de sécurité biologique à sécher. Étiqueter la lame avec un crayon sur le côté givré. </li></ol> 3. Fixation des échantillons sur les lames <ol><li> Préparer la solution de fixation en mélangeant 0,5 ml de formaldehyde à 37% 4,5 ml d&#39;éthanol à 99%. </li><li> Une fois que les lames sont séchées, sortir 2 ml de la solution de fixation fraîchement préparé et le verser sur le coulisseau de sorte que toute la surface de la glissière est couverte. </li><li> Laissez la solution sur la lame pendant 1 min. Rincer la lame pendant 1 min avec de l&#39;eau du robinet et laisser sécher à l&#39;air. </li></ol> 4. Faire la solution amylase pour le contrôle négatif <ol><li> Prendre 0,25 g de poudre d&#39;amylase et dissoudre dans 50 ml de dil&#39;eau calmé. Verser la solution dans un endroit propre bécher de 100 ml. </li><li> Immerger la lame dans le bécher de sorte que la moitié de la glissière reçoit le traitement et l&#39;autre moitié ne est pas traitée et ensuite incuber pendant 15 min à température ambiante (figure 1C). </li><li> Faire une note sur le côté de la glissière reçoit le traitement de l&#39;amylase. Tracez une ligne sur le dos de la lame indiquant la frontière entre le traitement et le contrôle. </li><li> Laver les lames avec ddH 2 O pour éliminer la solution d&#39;amylase et de laisser la lame sécher à l&#39;air. </li></ol> 5. Effectuez l&#39;acide périodique Schiff (PAS) Coloration et imagerie NOTE: PAS réactifs sont toxiques par inhalation et sont corrosifs, de sorte que les mesures doivent être fait dans une hotte chimique et les déchets doivent être correctement éliminés conformément aux directives institutionnelles. <ol><li> Placer la lame sur une surface plane et versez 1,50 à 2,00 ml de solution d&#39;acide périodique sur l&#39;échantillon. Incubate pendant 5 min à température ambiante. </li><li> Rincer les lames dans plusieurs charges d&#39;eau distillée. </li><li> Verser 1,50 à 2,00 ml de réactif de Schiff sur la lame et incuber à température ambiante pendant 15 min. </li><li> Laver la lame avec de l&#39;eau distillée pendant 5 minutes et laisser sécher à l&#39;air. </li><li> Appliquer 10 pi milieu de montage sur la lame et couvrir avec deux petites lamelles, ou d&#39;appliquer 50 pi et utiliser une grande lamelle. </li><li> Appliquer vernis à ongles transparent sur les bords de la lamelle, laisser sécher pendant la nuit. </li></ol> 6. obtenir des images au microscope optique binoculaire à l&#39;aide de l&#39;objectif 100X

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The authors have nothing to disclose.

Les étapes essentielles du présent article vidéo étaient au cours du lavage et de l&#39;amylase traitement des cellules. Alors que le lavage des lames, l&#39;étape clé utilisait une bouteille de lavage compressible plastique et laisser couler l&#39;eau doucement à travers l&#39;échantillon sur la lame et ne visant pas directement sur les échantillons. Même la pression d&#39;eau directe moindre causerait les cellules à venir de la lame. Une autre étape clé était d&#39;utiliser la même diapositive pendant ?...

L&#39;acide périodique Schiff (PAS) est une technique de coloration immunohistochimique qui est largement utilisé dans la recherche et le diagnostic musculaire. Il est également utilisé comme outil de diagnostic dans des échantillons de sang. La technique fonctionne en appliquant une solution d&#39;acide périodique à l&#39;échantillon, qui se oxyde à l&#39;intérieur des unités de polysaccharide créer des groupes aldéhyde qui réagissent avec le réactif de Schiff l&#39;incolore pour produire ainsi un produit de magenta foncé. Les étapes de cette procédure sont représentées sur la figure 1. La tache tourne avec quoi que ce soit, y compris des polysaccharides magenta glycogène, les glycoprotéines, les glycolipides, les mucines, ou d&#39;autres molécules ayant des groupements polysaccharidiques. Coloration PAS est souvent utilisé pour mesurer les niveaux de glycogène dans les fibres musculaires. Muscles sections de tissus sont idéales pour la technique qu&#39;ils attachent fermement à la diapositive et résister à de multiples étapes de lavage et de coloration. Le glycogène est plus présent dans contraction rapide de type II fibres musculaires, qui ont une forte demandepour la production d&#39;ATP rapide nécessitant glycogène pour 1,2 de performance maximale. Le glycogène est un polymère ramifié du glucose qui peut être divisé en glucose libre par l&#39;action d&#39;enzymes de la glycogène Phosphorylase. Dans les temps de repos et de suffisance nutritionnelle, le glycogène est reconstitué par le processus de glycogenèse, tandis qu&#39;en temps de l&#39;insuffisance ou de haute énergie nutritionnelle la demande; glycogène est décomposé en glucose par glycogénolyse. De dès les années 1950, les cliniciens-chercheurs ont exploré la coloration PAS sur des échantillons de sang pour analyser le contenu de glycogène dans diverses maladies 3-7. Par exemple, dans la maladie de Pompe, une glycogénose de bonne foi globules blancs aux maladies accumulent de grandes quantités de glycogène qui diffère sensiblement de 8 témoins sains. Cet article explique-vidéo d&#39;une version adaptée de coloration PAS pour une utilisation sur des cellules périphériques mononucléaires de sang périphérique (PBMC) des échantillons de sang veineux de sujets humains en bonne santé. PBMCs contient essentiellement des lymphocytes du lymphocyte T et les familles de lymphocytes B, ainsi que d&#39;autres cellules du système immunitaire telles que des cellules et des monocytes tueurs naturels. La première étape de purification élimine les erythrocytes, les neutrophiles, les granulocytes et d&#39;autres. Cette technique fournit des données sur une partie de concentré pour la numération des lymphocytes permettant plus robuste de cellules de PAS-positif par rapport à l&#39;aide de frottis de sang total. <img alt="Figure 1" src="/files/ftp_upload/52199/52199fig1highres.jpg" /> Figure 1:. Étape par la méthodologie de l&#39;étape de la coloration PAS sur CMSP (A) d&#39;abord, l&#39;isolement des PBMC est réalisé par gradient de ficoll, le panneau de gauche montre la préparation avant la centrifugation, le panneau de droite montre après centrifugation où la couche leucocytaire contenant les PBMC On observe dans le centre du tube. (B) PBMC isolées sont fixées sur la lame à l&#39;aide de formol-éthanol solu fixantstion. La lame est doucement rincé avec de l&#39;eau distillée dans une bouteille de lavage en matière plastique. (C) La lame est ensuite placé dans un bécher de 100 ml rempli à mi-chemin avec une solution d&#39;amylase, qui dissoudra glycogène. La lame est rincée doucement. (D) La lame est traité avec une solution d&#39;acide périodique, où l&#39;oxydation des saccharides a lieu. Les lames sont rincés doucement; cela va enlever l&#39;excès de l&#39;acide périodique et arrêter l&#39;étape d&#39;oxydation. (E) Lorsque le réactif de Schiff est ajouté aux lames, il va réagir avec des aldéhydes créés au cours de l&#39;étape d&#39;oxydation. Ce réactif incolore va alors donner un produit magenta rouge profond. Les lames sont doucement rincées pour éliminer le réactif Schiff excès.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Periodic Acid Shiff Kit</td>
			<td>Sigma-Aldrich</td>
			<td>395B</td>
			<td>Bring to room temperature prior to use. Materials in this kit are toxic and harmful. Use caution&#160; 
			<a href="http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/395b?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>&#945;-Amylase from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>A3176</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/a3176?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Binocular Microscope&#160;</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Axio Lab A0</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Assay Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK016</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&amp;region=CA">http://www.sigmaaldrich.com/catalog/product/sigma/mak016?lang=en&#38;region=CA</a></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>VWR, GE Healthcare</td>
			<td>17-1440-02</td>
			<td>Nonionic synthetic polymer of sucrose 
			<a href="https://us.vwr.com/store/catalog/product.jsp?product_id=4779441">https://us.vwr.com/store/catalog/product.jsp?product_id=4779441</a></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td>For PBMC isolation, swing buckets were used&#160;</td>
		</tr>
	</tbody>
</table>

detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Pour valider les réactifs et la technique de base, PAS coloration a été effectuée selon les instructions du fabricant sur des sections du muscle soléaire de souris. La coloration a été réalisée le même jour que le sacrifice et dans la dernière étape de la coloration, les coupes ont été fixées par du xylène. Le muscle soléaire est connu pour contenir ~ 35% de cellules de glycogène positif 9. Les cellules musculaires colorées affichent deux granulés ponctuées Caractéristiques- PAS-positifs...

Watch this Scientific Journal Video about Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining at JoVE.com

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Détection glycogène dans les cellules mononucléaires du sang périphérique avec l&#39;acide périodique Schiff coloration

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

detecting-glycogen-peripheral-blood-mononuclear-cells-with-periodic

Research

JoVE Journal

Immunology and Infection

21.0K vues.  Centre for Structural and Functional Genomics, PERFORM Centre, Concordia University. Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Vidéo: Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Induction de l&#39;alloantigène spécifique anergie dans les cellules mononucléaires du sang périphérique par stimulation alloantigène avec blocage du signal de co-stimulation

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Recherche

Immunologie et infection

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansion du sang périphérique humain cellules T γδ utilisant zolédronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Génération de cellules T humaines allo-spécifiques du sang périphérique

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

L&#39;isolement et la cytométrie en flux analyse des cellules mononucléaires du sang périphérique gliome infiltrant

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Analyse des Lymphocytes extravasation L&#39;utilisation d&#39;un<em&gt; In vitro</em&gt; Modèle de la barrière hématoencéphalique humaine

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Séparation des sous-populations de cellules immunitaires dans les échantillons de sang périphérique des enfants atteints de mononucléose infectieuse

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Isolement, activation et expansion des lymphocytes T par flottation à partir d’échantillons de cellules mononucléaires du sang périphérique humain à l’aide de microbulles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Collection et cryostockage PBMC pour l’analyse bioénergétique mitochondriale

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Pleins feux sur l’auteur : Préservation des paramètres bioénergétiques dans les cellules mononucléées du sang périphérique après cryoconservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

Cryoconservation et évaluation bioénergétique des cellules mononucléées du sang périphérique humain

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...