Nous sommes reconnaissants à Takaji Wakita et Jens Bukh pour JFH1 et les isolats de J6CF, respectivement. En outre, nous remercions Charles Rice pour Huh-7.5 des cellules et l&#39;anticorps 9E10, Steven Foung pour l&#39;anticorps spécifique à E2 CBH-23, et tous les membres du ministère de la virologie expérimentale, Twincore des suggestions utiles et des discussions.

<ol>
	<li>Zhu, Q., Guo, J. T., Seeger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+hepatitis+C+virus+subgenomes+in+nonhepatic+epithelial+and+mouse+hepatoma+cells.">Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells.</a> J. Virol. 77, 9204-9210 (2003).</li><li>Kato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonhepatic+cell+lines+HeLa+and+293+support+efficient+replication+of+the+hepatitis+C+virus+genotype+2a+subgenomic+replicon.">Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon.</a> J. Virol. 79, 592-596 (2005).</li><li>Ali, S., Pellerin, C., Lamarre, D., Kukolj, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+subgenomic+replicons+in+the+human+embryonic+kidney+293+cell+line.">Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line.</a> J. Virol. 78, 491-501 (2004).</li><li>Date, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+2a+hepatitis+C+virus+subgenomic+replicon+can+replicate+in+HepG2+and+IMY-N9+cells.">Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells.</a> J. Biol. Chem. 279, 22371-22376 (2004).</li><li>Chang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+hepatitis+C+virus+(HCV)+RNA+in+mouse+embryonic+fibroblasts:+protein+kinase+R+(PKR)-dependent+and+PKR-independent+mechanisms+for+controlling+HCV+RNA+replication+and+mediating+interferon+activities.">Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities.</a> J. Virol. 80, 7364-7374 (2006).</li><li>Zhong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+hepatitis+C+virus+infection+in+vitro.">Robust hepatitis C virus infection in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 9294-9299 (2005).</li><li>Lindenbach, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+replication+of+hepatitis+C+virus+in+cell+culture.">Complete replication of hepatitis C virus in cell culture.</a> Science. 309, 623-626 (2005).</li><li>Wakita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+infectious+hepatitis+C+virus+in+tissue+culture+from+a+cloned+viral+genome.">Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.</a> Nat. Med. 11, 791-796 (2005).</li><li>Witteveldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD81+is+dispensable+for+hepatitis+C+virus+cell-to-cell+transmission+in+hepatoma+cells.">CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells.</a> J. Gen. Virol. 90, 48-58 (2009).</li><li>Bitzegeio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+hepatitis+C+virus+to+mouse+CD81+permits+infection+of+mouse+cells+in+the+absence+of+human+entry+factors.">Adaptation of hepatitis C virus to mouse CD81 permits infection of mouse cells in the absence of human entry factors.</a> PLoS Pathog. 6, e1000978 (2010).</li><li>Lindemann, D., Goepfert, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+foamy+virus+envelope+glycoproteins.">The foamy virus envelope glycoproteins.</a> Curr Top Microbiol Immunol. 277, 111-129 (2003).</li><li>Brohm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+determinants+important+for+hepatitis+C+virus+p7+function+in+morphogenesis+by+using+trans-complementation.">Characterization of determinants important for hepatitis C virus p7 function in morphogenesis by using trans-complementation.</a> J. Virol. 83, 11682-11693 (2009).</li><li>Steinmann, E., Brohm, C., Kallis, S., Bartenschlager, R., Pietschmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+trans-encapsidation+of+hepatitis+C+virus+RNAs+into+infectious+virus-like+particles.">Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles.</a> J. Virol. 82, 7034-7046 (2008).</li><li>Koutsoudakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+early+steps+of+hepatitis+C+virus+infection+by+using+luciferase+reporter+viruses.">Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses.</a> J. Virol. 80, 5308-5320 (2006).</li><li>Frentzen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Completion+of+hepatitis+C+virus+replication+cycle+in+heterokaryons+excludes+dominant+restrictions+in+human+non-liver+and+mouse+liver+cell+lines.">Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.</a> PLoS Pathog. 7, e1002029 (2011).</li><li>Lindemann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+particle-associated+glycoprotein+signal+peptide+essential+for+virus+maturation+and+infectivity.">A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity.</a> J. Virol. 75, 5762-5771 (2001).</li><li>Blight, K. J., McKeating, J. A., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+permissive+cell+lines+for+subgenomic+and+genomic+hepatitis+C+virus+RNA+replication.">Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.</a> J. Virol. 76, 13001-13014 (2002).</li></ol>

TP a reçu des honoraires de consultation de Biotest.

Nous présentons deux méthodes pour induire la formation hétérocaryon dans des cellules cultivées pour l&#39;analyse des dominantes restrictions négatives qui empêchent la réplication du VHC. Utilisation ces procédures on exclut la présence d&#39;un élément dominant exprimé de manière constitutive ou induit par un virus dans différentes humaine non foie et dans des lignées cellulaires hépatiques de souris. Le premier essai analyse principalement si les facteurs de restriction de prévenir assemblage du V...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> DMEM </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> 41965-039 </td></tr><tr><td> L-glutamine </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> 25030-024 </td></tr><tr><td> Non-acides aminés essentiels </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> 11140-035 </td></tr><tr><td> Pénicilline / streptomycine </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> 15140-122 </td></tr><tr><td> Sérum de veau foetal </td><td> AAP, Cölbe, Allemagne </td><td> A15151 </td></tr><tr><td> α-E2 (CBH23) </td><td> aimablement fournies par Steven Foung 10 </td><td></td></tr><tr><td> ATP </td><td> Sigma, Steinheim, Allemagne </td><td> A2833-106 </td></tr><tr><td> Le glutathion </td><td> Sigma, Steinheim, Allemagne </td><td&gt; G4251-1G </td></tr><tr><td> Blasticidine </td><td> Invivo Gen, San Diego, Etats-Unis </td><td> Ant-bl-1 </td></tr><tr><td> G418 (généticine) </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> 11811-064 </td></tr><tr><td> Polyéthylène-glycol-1500 </td><td> Roche, Mannheim, Allemagne </td><td> 10783641001 </td></tr><tr><td> Paraformaldéhyde </td><td> Roth, Karlsruhe, Allemagne </td><td> 0335,3 </td></tr><tr><td> Triton X-100 </td><td> Roth, Karlsruhe, Allemagne </td><td> 3051,2 </td></tr><tr><td> Du sérum de chèvre </td><td> Sigma, Steinheim, Allemagne </td><td> G9023-5mL </td></tr><tr><td> α-NS5A (9E10) </td><td> Aimablement communiqué par Charles Rice 7 </td><td></td></tr><tr><td> DAPI (4 &#39;, 6&#39;-diamidino-2-phénylindole dichlorhydrate) </td><td> Invitrogen </td><td> D1306 </td></tr><tr><td> Alexa Fluor-546 - chèvre anti-IgG humaine </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> A21089 </td&gt; </tr><tr><td> Alexa Fluor-488 - chèvre anti-IgG de souris </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> A10680 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> 11668-019 </td></tr><tr><td> CellTracker CMTMR </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> C2927 </td></tr><tr><td> CellTracker CMFDA </td><td> Invitrogen, Karlsruhe, Allemagne </td><td> C2925 </td></tr><tr><td> Fluoromount </td><td> Sigma, Steinheim, Allemagne </td><td> F4680-25ML </td></tr><tr><td> Tous les autres produits chimiques </td><td> Roth, Karlsruhe, Allemagne </td><td></td></tr><tr><td> Matériaux de culture cellulaire </td><td> Sarstedt, Nümbrecht, Allemagne </td><td></td></tr></tbody></table>

two methods of heterokaryon formation to discover hcv restriction factors

L&#39;hépatite C (VHC) est un virus hépatotrope avec une gamme d&#39;hôtes limitée pour les humains et les chimpanzés. Bien que l&#39;ARN du VHC de réplication a été observée dans les lignées cellulaires humaines non-hépatiques et murin, l&#39;efficacité était très faible et les procédures de sélection requis à long terme en utilisant le VHC réplicon construit exprimant dominantes antibiotiques sélectionnables marqueurs 1-5. VHC in vitro de la recherche se limite donc à des lignées cellulaires d&#39;hépatome permissives pour l&#39;entrée du virus et l&#39;achèvement du cycle de vie virale. En raison de HVC tropisme espèces étroit, il n&#39;ya pas de modèle animal immunocompétent petite disponibles qui soutient le cycle de réplication du VHC complète 6-8. La réplication du VHC dans inefficace des cellules non humaines, par exemple d&#39;origine de la souris est probablement dû à un manque d&#39;incompatibilité génétique des facteurs essentiels de dépendance d&#39;accueil et / ou l&#39;expression de facteurs de restriction. Nous avons étudié si la propagation du VHC est supprimée par fa restriction dominantectors en soit des lignées cellulaires humaines dérivées de non-hépatiques tissus ou dans des lignées de souris cellules hépatiques. À cette fin, nous avons développé deux indépendants conditionnelles trans-complémentation des méthodes reposant sur ​​la fusion de cellules somatiques. Dans les deux cas, l&#39;achèvement du cycle de réplication virale n&#39;est possible que dans les hétérocaryons. Par conséquent, le succès trans-complémentation, qui est déterminée en mesurant la production de novo de la descendance virale infectieuse, indique l&#39;absence de restrictions dominantes. Plus précisément, subgénomiques réplicons du VHC portant un transgène luciférase ont été transfectés dans des cellules permissives hautement hépatome humain (Huh-7.5 des cellules). Par la suite, ces cellules ont été co-cultivées et fusionné à diverses cellules humaines et murines exprimant VHC noyau structurel protéines, de l&#39;enveloppe 1 et 2 (E1, E2) et accessoire protéines p7 et NS2. À condition que la fusion cellulaire a été initiée par un traitement avec du polyéthylène-glycol (PEG), la culture virale infectieuse libérée particles qui a infecté des cellules naïves d&#39;une manière dépendante du récepteur. Pour évaluer l&#39;influence des restrictions dominantes sur le cycle de vie complet, y compris virale entrée dans la cellule, la traduction d&#39;ARN, la réplication et l&#39;assemblage du virus, nous avons profité d&#39;une lignée cellulaire de foie humain (Huh-7 Lunet N cellules 9) qui manque de l&#39;expression endogène de CD81, un facteur essentiel de l&#39;entrée du VHC. En l&#39;absence de CD81 exprimés de manière ectopique, ces cellules sont essentiellement réfractaire à l&#39;infection par le VHC 10. Il est important, en cas de co-culture et fusionnées avec des cellules qui expriment CD81 humain, mais le manque au moins un autre facteur crucial entrée dans la cellule (c.-à-SR-BI, CLDN1, OCLN), seuls les hétérocaryons résultant d&#39;afficher l&#39;ensemble des facteurs d&#39;entrée du VHC nécessaires pour l&#39;infection. Par conséquent, pour analyser si les facteurs de restriction dominantes supprimer l&#39;achèvement du cycle de réplication du VHC, on condensé Lunet N cellules avec des cellules d&#39;origine humaine différents et une souris qui remplissent la critique mentionnée ci-dessuseria. Lorsque des cellules co-cultivées ont été transfectées avec une protéine d&#39;enveloppe virale hautement fusogène mutante de type foamy virus du prototype (PFV 11) et par la suite avec des particules de VHC défi infectieuses HCVcc), de novo de production de virus infectieux a été observée. Cela indique que le VHC complété avec succès son cycle de réplication dans les hétérocaryons écartant ainsi l&#39;expression de facteurs de restriction dominantes dans ces lignées cellulaires. Ces nouveaux conditionnelles trans-complémentation méthodes sera utile pour dépister un large panel de lignées cellulaires et cellules primaires pour l&#39;expression du VHC-spécifiques facteurs de restriction dominantes.

Watch this Scientific Journal Video about Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors at JoVE.com

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Nous décrivons deux méthodes pour conditionnelle<em&gt; Trans</em&gt;-Complémentation de l&#39;hépatite C (VHC) de montage et la réalisation du cycle de vie virale complète, qui reposent sur la formation des hétérocaryons. Ces techniques sont adaptées à l&#39;écran pour les lignées cellulaires qui expriment des facteurs de restriction dominantes, qui empêchent la production de la descendance du VHC infectieux.

Deux méthodes de formation hétérocaryons à découvrir les facteurs de restriction du VHC

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

two-methods-heterokaryon-formation-to-discover-hcv-restriction

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JoVE Journal

Immunology and Infection

11.5K vues.  Twincore, Centre for Experimental and Clinical Infection Research. Nous décrivons deux méthodes pour conditionnelle Trans-Complémentation de l'hépatite C (VHC) de montage et la réalisation du cycle de vie virale complète, qui reposent sur la formation des hétérocaryons. Ces techniques sont adaptées à l'écran pour les lignées cellulaires qui expriment des facteurs de restriction dominantes, qui empêchent la production de la descendance du VHC infectieux.

Vidéo: Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Méthodes d&#39;évaluation bêta Mediated la mort cellulaire par les lymphocytes T cytotoxiques

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Recherche

Immunologie et infection

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

Une enzyme de restriction basée sur le clonage méthode pour évaluer la<em&gt; In vitro</em&gt; Capacité de réplication du VIH-1 sous-type C Gag-MJ4 chimériques virus

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Création et optimisation d&#39;une installation à haut débit pour l&#39;étude<em&gt; Staphylococcus epidermidis</em&gt; Et<em&gt; Mycobacterium marinum</em&gt; Infection comme un modèle pour la découverte de médicaments

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

Méthodes pour évaluer la cytotoxicité et l&#39;immunosuppression des préparations de produits combustibles tabac

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

À long terme intravitale microscopie multiphotonique imagerie des cellules immunitaires dans sains et malades du foie en utilisant des souris CXCR6.Gfp Reporter

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Méthodes pour augmenter la sensibilité de la haute résolution de fusion Single Nucleotide Polymorphism génotypage dans le paludisme

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

L&#39;application d&#39;un système d&#39;expression inductible pour étudier les interférences de facteurs de virulence bactérienne avec signalisation intracellulaire

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Méthodes pour inhiber bactérienne Pyomelanin production et de déterminer l&#39;augmentation correspondante de la sensibilité au stress oxydatif

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Décrypter et de l&#39;imagerie et la pathogenèse de Cording<em&gt; Mycobacterium abscessus</em&gt; Dans embryons de poissons zèbres

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

Méthodes pour étudier le rôle de réglementation de petits ARN ribosomal et l&#39;occupation des<em&gt; Plasmodium falciparum</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...