Die Autoren bedanken sich bei Anna Walduck, Christina Cheers, Stuart Berzins, Dale Godfrey, Yifan Zhan und Jonathan Wilksch für Beratung und Reagenzien zu danken. Diese Arbeit wurde von der Juvenile Diabetes Research Foundation (1-2008-602) und die Australian National Health and Medical Research Council (575552) gefördert. NW wird von einem Australian Postgraduate Award unterstützt. OW wird von einer RD Wright Fellowship von der Australian National Health Medical Research Council unterstützt.

<ol>
	<li>Wing, E. J., Gregory, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes:+clinical+and+experimental+update.">Listeria monocytogenes: clinical and experimental update.</a> J Infect Dis. 185, S18-S24 (2002).</li><li>Conlan, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+host-pathogen+interactions+in+the+liver+and+spleen+during+systemic+murine+listeriosis:+an+overview.">Early host-pathogen interactions in the liver and spleen during systemic murine listeriosis: an overview.</a> Immunobiology. 201, 178-187 (1999).</li><li>Neuenhahn, M., Busch, D. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+and+susceptibility+of+mice+to+bacterial+infection:+genetics+of+listeriosis.">Resistance and susceptibility of mice to bacterial infection: genetics of listeriosis.</a> Infect Immun. 19, 755-762 (1978).</li><li>Garifulin, O., Boyartchuk, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Listeria+monocytogenes+as+a+probe+of+immune+function.">Listeria monocytogenes as a probe of immune function.</a> Brief Funct Genomic Proteomic. 4, 258-269 (2005).</li><li>Gervais, F., Stevenson, M., Skamene, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+resistance+to+Listeria+monocytogenes:+regulation+of+leukocyte+inflammatory+responses+by+the+Hc+locus.">Genetic control of resistance to Listeria monocytogenes: regulation of leukocyte inflammatory responses by the Hc locus.</a> J Immunol. 132, 2078-2083 (1984).</li><li>Gervais, F., Desforges, C., Skamene, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C5-sufficient+A/J+congenic+mouse+strain.+Inflammatory+response+and+resistance+to+Listeria+monocytogenes.">The C5-sufficient A/J congenic mouse strain. Inflammatory response and resistance to Listeria monocytogenes.</a> J Immunol. 142, 2057-2060 (1989).</li><li>Boyartchuk, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multigenic+control+of+Listeria+monocytogenes+susceptibility+in+mice.">Multigenic control of Listeria monocytogenes susceptibility in mice.</a> Nat Genet. 27, 259-260 (2001).</li><li>Boyartchuk, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+resistance+locus+sst1+controls+innate+immunity+to+Listeria+monocytogenes+infection+in+immunodeficient+mice.">The host resistance locus sst1 controls innate immunity to Listeria monocytogenes infection in immunodeficient mice.</a> J Immunol. 173, 5112-5120 (2004).</li><li>Pan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ipr1+gene+mediates+innate+immunity+to+tuberculosis.">Ipr1 gene mediates innate immunity to tuberculosis.</a> Nature. 434, 767-772 (2005).</li><li>Garifulin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irf3+polymorphism+alters+induction+of+interferon+beta+in+response+to+Listeria+monocytogenes+infection.">Irf3 polymorphism alters induction of interferon beta in response to Listeria monocytogenes infection.</a> PLoS Genet. 3, 1587-1597 (2007).</li><li>Hof, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+different+strains+of+Listeria+monocytogenes+serovar+1/2a.">Virulence of different strains of Listeria monocytogenes serovar 1/2a.</a> Med Microbiol Immunol (Berl. 173, 207-218 (1984).</li><li>Wollert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+the+host+range+of+Listeria+monocytogenes+by+rational+protein+design.">Extending the host range of Listeria monocytogenes by rational protein design.</a> Cell. 129, 891-902 (2007).</li><li>Lecuit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+model+for+listeriosis:+role+of+internalin+in+crossing+the+intestinal+barrier.">A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.</a> Science. 292, 1722-1725 (2001).</li><li>Marco, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+Listeria+monocytogenes+in+mice+infected+by+the+oral+route.">Penetration of Listeria monocytogenes in mice infected by the oral route.</a> Microb Pathog. 23, 255-263 (1997).</li><li>Busch, D. H., Vijh, S., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+model+for+infection+with+Listeria+monocytogenes.">Animal model for infection with Listeria monocytogenes.</a> Curr Protoc Immunol. Chapter. 19, 19-19 (2001).</li><li>Neuenhahn, M., Schiemann, M., Busch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+in+mouse+models+of+intracellular+bacterial+infection.">DCs in mouse models of intracellular bacterial infection.</a> Methods Mol Biol. 595, 319-329 (2010).</li><li>Conlan, J. W., North, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+are+essential+for+early+anti-Listeria+defense+in+the+liver,+but+not+in+the+spleen+or+peritoneal+cavity,+as+revealed+by+a+granulocyte-depleting+monoclonal+antibody.">Neutrophils are essential for early anti-Listeria defense in the liver, but not in the spleen or peritoneal cavity, as revealed by a granulocyte-depleting monoclonal antibody.</a> J Exp Med. 179, 259-268 (1994).</li><li>Czuprynski, C. J., Brown, J. F., Wagner, R. D., Steinberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+antigranulocyte+monoclonal+antibody+RB6-8C5+prevents+expression+of+acquired+resistance+to+Listeria+monocytogenes+infection+in+previously+immunized+mice.">Administration of antigranulocyte monoclonal antibody RB6-8C5 prevents expression of acquired resistance to Listeria monocytogenes infection in previously immunized mice.</a> Infect Immun. 62, 5161-5163 (1994).</li></ol>

Keine Interessenskonflikte erklärt.

Listeria is one of the most widely used organisms to characterize host immune responses to intracellular bacteria6. The protocol presented here enables one to measure bacterial load and immune cell responses within the same tissue of a given mouse. This dual measurement of a particular tissue for each infected mouse provides for more robust comparisons within and between mouse cohorts (either representing different mouse strains or time points post-infection). While Listeria infection by oral administration could also be used to study immune responses in mice, infection by intravenous injection is often used because: 1) it ensures rapid and effective delivery to the bloodstream; 2) it results in a synchronized and consistent systemic infection; and 3) the Mus species harbors a mutation in the gene encoding the E-cadherin receptor, which limits Listeria infection by oral administration (this mutation affects Listeria's ability to bind the mouse E-cadherin receptor and to efficiently cross the epithelial lining of the gastrointestinal tract)16-18.
There are a few critical steps in this protocol. First, the Listeria inoculum stock should be generated from a fresh overnight HBA culture to ensure viability and virulence. Second, it is important to accurately determine the CFU concentration of the inoculum before and after injecting all mice to ensure that the CFU concentration does not differ greatly between the first and last mouse injected. Third, injection of Listeria into the tail vein must be consistent for all mice. Lastly, it is necessary to perfuse the liver to deplete non-resident leukocytes to ensure accurate measurement of immune cells within the liver and not leukocytes passing through in the peripheral blood. All of these steps, if not performed successfully, can result in unwanted variability for bacterial load and/or immune responses between individual mice infected with Listeria. 
Two limitations of this protocol are the investigator's skill for infecting mice by intravenous injection and the detection of bacterial load in tissues. If one person is injecting a large number of mice, Listeria viability (i.e. CFU concentration) may reduce over time once the frozen inoculum is thawed (e.g. &gt;2 hours between injecting first and last mouse). It is up to the investigator to determine how many mice she/he can inject before the inoculum is compromised. Another limitation of this method is that the Listeria load cannot be accurately measured below 100CFU/organ due to the relatively small amounts of cultured tissue homogenate (e.g. Figure 3 indicates that 100CFU/organ is the detection limit). To more accurately measure lower values for CFU/organ, a larger amount of the tissue homogenate can be cultured (up to 0.5mL per plate, multiple plates can be used) so a greater proportion of the tissue is sampled for detection of Listeria. If a Stomacher is not available, then alternative methods to homogenate the tissue, such as using a tissue homogenizer, can be used instead for steps 7.1-7.2. On a practical note, if space in the 37&deg;C incubator is limited for culturing tissue samples from infected mice, then HBA culture plates can be incubated at room temperature for 2-3 days (the colonies will grow slower at room temperature). However, room temperature should not be used when growing cultures for preparation of frozen Listeria stocks.
This protocol provides a basic approach for characterizing Listeria infection in mice and can be used with other Listeria strains besides EGD. In addition to the liver and spleen, single-cell suspensions from lymph nodes can also be generated. In either instance, these single-cell suspensions can be used for a variety of analyses, including measuring immune cell subsets, and in vitro stimulation of sorted immune cells. Once the basic techniques are mastered this protocol can also be modified to isolate Listeria-specific T cells19, characterize dendritic cells20, or perform immune cell depletion at certain time-points after infection21,22 to more thoroughly characterize the immune response in mice infected with Listeria.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> Pferd Blut-Agar-Basis No.2 </td><td> Oxoid </td><td> CM0271 </td><td> Vorbereitung der Agar-Basis für HBA-Platten </td></tr><tr><td> Pferd Blut (defibriniertes) </td><td> Oxoid </td><td> HB1000 </td><td> Fügen Sie 5-10% auf Agar-Basis </td></tr><tr><td> Brain Heart Infusion (BHI) Brühe (10 mL) </td><td> Oxoid </td><td> TM456 </td><td></td></tr><tr><td> Brain Heart Infusion dehydrierte Medien </td><td> Oxoid </td><td> CM1135 </td><td></td></tr><tr><td> 96-well Flachboden Platte </td><td> BD Biosciences </td><td> 353072 </td><td></td></tr><tr><td> 70 um Zellsieb </td><td> BD Biosciences </td><td> 352350 </td><td></td></tr><tr><td> Kleine Petrischale </td><td> BD Biosciences </td><td> 351007 </td><td></td></tr><tr><td> Stomacher 80 Biomaster Lab-System </td><td> Seward </td><td></td><td></td></tr><tr><td> Plastic Stomacherbeuteln </td><td> Sarstedt </td><td> 86 9924 530 </td><td></td></tr><tr><td> Rinderserumalbumin </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> FACS-Puffer </td><td></td><td></td><td> 0,1% (w / v) Rinderserumalbumin in PBS </td></tr><tr><td> Isotonischen Percoll (33,75% Percoll in PBS) </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> 33.75mL Percoll, 3.75mL 10x PBS, macht 62.5mL 1x PBS 100ml isotonischer Percoll </td></tr><tr><td> TAC Buffer </td><td></td><td></td><td> 17mm Tris, 140mm Ammoniumchlorid in destilliertem Wasser </td></tr><tr><td> FCS / EDTA-Puffer </td><td></td><td></td><td> Fötalem Kälberserum mit 10mm EDTA </td></tr><tr><td> FACS / EDTA-Puffer </td><td></td><td></td><td> FACS-Puffer + 5 mM EDTA </td></tr><tr><td> Trypanblau </td><td> Sigma </td><td> T6146 </td><td> 0,4% (w / v) in PBS, sterilfiltriert </td></tr><tr><td> 2 ml-Kryoröhrchen </td><td> Greiner Bio One </td><td> 121263 </td><td></td></tr><tr><td> 27 gauge/1mL Insulinspritze </td><td> Terumo Medical Products </td><td> SS10M2713 </td><td></td></tr><tr><td> Nadel </td><td> Terumo Medical Products </td><td> NN-2516R (25G 5/8in) NN-2613R (26G 1/2in) </td><td></td></tr><tr><td> Spritze </td><td> Terumo Medical Produkte </td><td> SS-01T (1 mL), SS-053 (5 mL), SS-10S (10 mL) </td><td></td></tr></tbody></table>

measuring bacterial load and immune responses in mice infected with listeria monocytogenes

Listeria monocytogenes (Listerien) ist ein Gram-positive fakultativ intrazellulären Erregers 1. Maus-Studien typischerweise intravenöser Injektion von Listeria, was zu einer systemischen Infektion 2. Nach der Injektion Listeria schnell verbreitet, die Milz und Leber durch die Aufnahme durch CD8α + dendritischen Zellen und Kupffer-Zellen 3,4. Sobald phagozytiert, ermöglichen verschiedene bakterielle Proteine ​​Listeria dem Phagosom entkommen, überleben im Cytosol und infizieren benachbarte Zellen 5. In den ersten drei Tagen der Infektion, vermitteln verschiedene angeborene Immunsystem-Zellen (zB Monozyten, Neutrophilen, NK-Zellen, dendritische Zellen) bakterizide Mechanismen, Listeria Verbreitung zu minimieren. CD8 + T-Zellen werden anschließend rekrutiert und für den eventuellen Clearance von Listerien aus dem Host, in der Regel innerhalb von 10 Tagen der Infektion 6. Erfolgreiche Clearance von Listerien aus infizierten Mäusen hängt von der entsprechenden Auftreten von Host Immunantwort 6. Es gibt ein breites Spektrum an Empfindlichkeiten unter Inzucht-Mausstämmen 7,8. Generell sind Mäuse mit einer erhöhten Anfälligkeit für Listeria Infektion weniger in der Lage, bakterielle Vermehrung steuern, zeigen erhöhte bakterielle Belastung und / oder verzögerte Clearance im Vergleich zu resistenten Mäusen. Genetische Studien, einschließlich Kopplungsanalyse und Knockout-Maus-Stämme wurden verschiedene Gene für die Sequenzvariation betrifft Host Reaktionen auf Listeria Infektion 6,8-14 identifiziert. Ermittlung und Vergleich der Infektion Kinetik zwischen verschiedenen Mausstämmen ist daher eine wichtige Methode zur Identifizierung von Host-genetischen Faktoren, die die Immunantwort gegen Listeria beitragen. Vergleich der Host-Reaktionen auf verschiedene Listeria-Stämme ist auch ein effektiver Weg zur bakteriellen Virulenzfaktoren, die als potenzielle Ziele für Antibiotika-Therapie oder Impfstoff-Design dienen könnten, zu erkennen. Wir beschreiben hier eine einfache Methode zur Messung der Keimbelastung (Kolonie bildende Einheiten [KBE] pro Gewebe) und die Vorbereitung Single-Cell-Suspensionen der Leber und der Milz für FACS-Analyse von Immunantworten in Listeria-infizierten Mäusen. Diese Methode ist besonders nützlich für die erstmalige Charakterisierung von Listeria-Infektionen bei neuartigen Maus-Stämme, sowie Vergleich der Immunantworten zwischen verschiedenen Mausstämmen mit Listeria infiziert. Wir nutzen die Listeria monocytogenes EGD-Stamm 15, die, wenn sie auf Blutagar kultiviert, weist eine charakteristische halo-Zone um jede Kolonie durch β-Hämolyse 1 (Abbildung 1). Bakterielle Belastung und Immunreaktionen können jederzeit-Punkt nach der Infektion durch Kultivierung Gewebehomogenat auf Blutagarplatten und Vorbereitung Gewebe Zellsuspensionen für FACS-Analyse unter Verwendung der Protokolle im Folgenden beschrieben wird. Wir möchte darauf hinweisen, dass Personen, die immungeschwächte oder schwanger sind, sollten nicht mit Listeria, und die einschlägigen institutionellen Kommission für Biosicherheit und Tier Facility Management sollte konsultiert werden, bevor die Arbeit beginnt werden.

Watch this Scientific Journal Video about Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes at JoVE.com

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes ist ein Modellorganismus für die Untersuchung Immunreaktionen und die genetische Anfälligkeit für Bakterien bei Mäusen intrazellulär. Diese Methode ermöglicht ein bis bakterielle Belastung zu messen und generieren Single-Cell-Suspensionen der Leber und Milz von Mäusen für FACS-Analyse auf Veränderungen in Immunzellen durch Listeria-Infektion zu bestimmen.

Mess-bakterielle Belastung und Immunreaktionen in Mäuse Infizierte mit Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

measuring-bacterial-load-immune-responses-mice-infected-with-listeria

Research

JoVE Journal

Immunology and Infection

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

30.8K Aufrufe.  The University of Melbourne. Listeria monocytogenes ist ein Modellorganismus für die Untersuchung Immunreaktionen und die genetische Anfälligkeit für Bakterien bei Mäusen intrazellulär. Diese Methode ermöglicht ein bis bakterielle Belastung zu messen und generieren Single-Cell-Suspensionen der Leber und Milz von Mäusen für FACS-Analyse auf Veränderungen in Immunzellen durch Listeria-Infektion zu bestimmen.

Serie: Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Mess-Calpain Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Forschung

Immunologie und Infektion

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Mündliche Übertragung von<em&gt; Listeria monocytogenes</em&gt; In Mäusen über die Aufnahme von kontaminierten Speisen

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Imaging InlC Sekretion to Cellular-Infektion durch den bakteriellen Erreger untersuchen<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Beurteilung der bakteriellen Invasion von Herzzellen in Kultur und Herzkolonisation bei infizierten Mäusen mit Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

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

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

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

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

Ein<em&gt; In-vitro-</em&gt; Modell zur Messung der Immunantwort auf Malaria im Kontext der HIV-Koinfektion

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

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA-Reinigung aus intrazellulär Grown<em&gt; Listeria monocytogenes</em&gt; In Makrophagenzellen

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

Experimental Infection with &lt;em&gt;Listeria monocytogenes&lt;/em&gt; as a Model for Studying Host Interferon-&amp;#947; Responses

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Experimentelle Infektion mit<em&gt; Listeria monocytogenes</em&gt; Als Modell für die Untersuchung Host-Interferon-γ Antworten

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Bewertung der Zika-Virus-spezifischen T-Zell-Reaktionen in Organe der Immunabwehr von infizierten Ifnar1- / - Mäusen

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Listeria monocytogenes Infection of the Brain

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. monocytogenes to cross the blood-brain barrier (BBB) is concerning as it can lead to life-threatening meningitis and encephalitis. The BBB protects the brain microenvironment from various toxic metabolites and microbial pathogens found in the blood following infection, and therefore supports brain homeostasis. The mechanisms by which L. monocytogenes present in the bloodstream cross the BBB to cause brain infections are not fully understood and there is also a lack of a robust model system to study brain infections by L. monocytogenes. Here, we present a simple mouse infection model to determine whether bacteria have crossed the BBB and to quantitate the burden of bacteria that have colonized the brain in vivo. In this method, animals were infected intravenously with L. monocytogenes and were humanely euthanized by exposure to CO2 followed by cervical dislocation. Cardiac perfusion of the animals was performed prior to harvesting infected organs. Blood was collected before perfusion and the number of bacteria per organ or mL of blood was determined by plating dilutions of the blood or organ homogenates on agar plates and counting the number of colonies formed. This method can be used to study novel receptor-ligand interactions that enhance infection of the brain by L. monocytogenes and can be easily adapted for the study of multiple bacterial pathogens.

Listeria monocytogenes Infection of the Brain

During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided.

Listeria monocytogenes Infektion des Gehirns

Listeria monocytogenes is an intracellular bacterial pathogen that is frequently associated with food-borne infection. The ability of&#160;L. ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Bildgebung der In Situ Interferon Gamma Produktion in der Maus Milz nach Listeria monocytogenes Infektion

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...