Development of this protocol was supported by an operating grant from CIHR (MOP97807) to SED.

No conflicts of interest to disclose.

Qui si descrive un protocollo di come effettuare una infezione sperimentale di base con il ceppo EGD di L. monocytogenes 25 in C57BL 6J maschi o femmine /. Questo protocollo è stato istituito con lo scopo di studiare l&#39;effetto di un romanzo piccola molecola IS001 sulla produzione di IFN-γ da parte dei linfociti innata e adattativa in vivo 31. Monitorando la clearance batterica e la sopravvivenza dopo l&#39;infezione, intuizioni sono state acquisite in modo questi cambiamenti...

IFN-γ è una citochina che è cruciale per mediare l&#39;immunità contro i patogeni intracellulari e per il controllo della crescita tumorale 5. L&#39;importanza di questa citochina nella resistenza batterica è evidente nell&#39;osservazione che gli esseri umani con mutazioni nel percorso di segnalazione di IFN-γ sono altamente suscettibili all&#39;infezione con micobatteri e salmonelle 6. Allo stesso modo, i topi carenti di entrambi IFN-γ o difetti del recettore espositive IFN-γ nella resistenza alla micobatteri 7-9 e altri agenti patogeni intracellulari, tra cui L. monocytogenes 10,11, Leishmania importante 12, Salmonella typhimurium 13, e alcuni virus 11. Oltre agli agenti patogeni Lotta contro, IFN-γ gioca un ruolo cruciale in Host-difesa contro i tumori 14. Sebbene maggiore produzione di IFN-γ è utile nel contesto di infezione o tumore, la produzione prolungata di questa citochina è stato collegato to lo sviluppo di autoimmunità sistemica 15-17 e l&#39;accelerazione del diabete di tipo I nei diabetici non obesi modello di topo 18. Le principali fonti di IFN-γ includono le cellule NK, cellule NKT, γδ cellule T helper, le cellule T 1 (Th1), e linfociti T citotossici (CTL) 5,19,20. IFN-γ migliora sia innata e adattativa per: (1) up-regolazione complesso maggiore di istocompatibilità (MHC) classe I e II espressione, (2) aumentare l&#39;espressione di molecole co-stimolatorie sulle cellule presentanti l&#39;antigene, (3) macrofagi migliorare fattori fagocitosi e la produzione di citochine pro-infiammatorie e microbicidi (ad esempio, l&#39;ossido di azoto e le specie reattive dell&#39;ossigeno), (4) promuovere la differenziazione delle cellule T CD4 + naive in cellule effettrici Th1, (5) promuovere il passaggio di classe di anticorpi di immunoglobuline ( Ig) 2a e IgG3 (nel topo), (6) inducendo la produzione di chemochine reclutare cellule immunitarie di siti di infection, e (7) miglioramento delle cellule NK e le risposte CTL 5,19. Data l&#39;importanza cruciale di IFN-γ in risposta dell&#39;ospite agli agenti patogeni e tumori, ricombinante IFN-γ è stato testato come trattamento per varie infezioni e neoplasie (recensito in 19). Tuttavia, a causa somministrazione sistemica di IFN-γ o citochina Th1 promuovere l&#39;interleuchina-12 (IL-12) è associato ad effetti collaterali e dose-correlati tossicità 19,21, vi è interesse a sviluppare strategie alternative per aumentare la produzione di IFN-γ by cellule immunitarie. Sviluppo di nuovi farmaci biologici e piccole molecole richiede in strumenti di screening vivo per verificare se tali agenti aumentare la produzione di IFN-γ durante una risposta immunitaria e se questo si traduce in effetti biologici significativi come l&#39;aumento della sopravvivenza degli animali. Infezione sperimentale di topi con i gram-positivi batterio L. monocytogenes è stato un modello strumentale perdecifrare il ruolo di IFN-γ in host-immunità contro patogeni intracellulari 1,22. L&#39;infezione dei topi con l&#39;agente patogeno per via endovenosa o per via intraperitoneale (ip) porta alla rapida diffusione dei batteri alla milza e nel fegato, dove diventano internalizzati dai macrofagi residenti e epatociti con cariche batteriche picco nella milza che si verificano tra il 3 e 4 giorni post infezione 1,3,22. La produzione di IFN-γ da parte delle cellule NK è importante per l&#39;attivazione dei macrofagi e la resistenza precoce contro l&#39;agente patogeno 3; tuttavia a dosi elevate infettive, la produzione di IFN-γ può anche essere dannoso alla clearance dei patogeni 23. Cellule NKT sono anche una fonte di IFN-γ nella milza e nel fegato durante il controllo precoce di agenti patogeni 2,24 e questa produzione è stato dimostrato per amplificare la produzione di IFN-γ da altri tipi di cellule, tra cui le cellule NK 2. D&#39;altra parte, in seguito ad azione adattativi linfociti T, cellule T CD8 + in partilare, sono importanti per mediare il gioco del patogeno e della protezione contro reinfezione 1,4,22. Questo modello di infezione è stato interessante per i ricercatori per una serie di ragioni (rivisto in 1). Innanzitutto, l&#39;infezione con il patogeno è altamente riproducibile e induce una forte Th1 e cellulare risposta immunitaria. In secondo luogo, durante l&#39;infezione subletali, carica batterica è concentrata nel fegato e nella milza dove può essere facilmente misurata. In terzo luogo, l&#39;agente patogeno può essere tranquillamente gestita sotto biosicurezza di livello 2 (BSL2) condizioni. In quarto luogo, l&#39;organismo e la risposta immunitaria che genera sono stati ampiamente caratterizzati. Infine, una varietà di ceppi mutanti e geneticamente modificati sono stati sviluppati che sono disponibili per l&#39;uso. Qui descritto è un protocollo di base per l&#39;inoculazione di C57BL 6J topi / con il ceppo EGD di L. monocytogenes 25 e per la misura di IFN - γ resposte da NK, NKT e linfociti adattivi post-infezione. Inoltre descritto è come misurare carica batterica nella milza e nel fegato dopo l&#39;infezione subletali e di effettuare studi di sopravvivenza dopo infezione con un modificata LD 50 dose del patogeno. Infine, dati rappresentativi sono mostrati di come questo protocollo può essere usata per selezionare l&#39;effetto di nuovi trattamenti sulle risposte IFN-gamma e la sopravvivenza mouse dalla L. monocytogenes infezione.

<table><tbody><tr><td>Brain Heart Infusion Broth, Modified</td><td>BD</td><td>299070</td><td>any brand should be appropriate</td></tr><tr><td>Agar</td><td>BD</td><td>214010</td><td>any brand should be appropriate</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td>any brand should be appropriate</td></tr><tr><td>1x PBS</td><td>Sigma</td><td>D8537</td><td>any brand should be appropriate</td></tr><tr><td>TissueLyser II</td><td>Qiagen</td><td>85300</td><td>any brand should be appropriate</td></tr><tr><td>Ammonium Chloride (NH&lt;sub&gt;3&lt;/sub&gt;Cl)</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>22400089</td><td>any brand should be appropriate</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco</td><td>12483</td><td>Before use, heat-inactivate at 56 &amp;deg;C for 30 min</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030</td><td>any brand should be appropriate</td></tr><tr><td>Non-essential amino acids</td><td>Gibco</td><td>11140</td><td>any brand should be appropriate</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td>any brand should be appropriate</td></tr><tr><td>GolgiStop Protein Transport Inhibitor (containing Monensin)</td><td>BD</td><td>554724</td><td>Use 4 &amp;mu;l in 6 ml cell culture</td></tr><tr><td>16% Paraformaldehye</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Dilute to 4% PFA in ddH&lt;sub&gt;2&lt;/sub&gt;O or 1x PBS</td></tr><tr><td>10x Perm/Wash buffer</td><td>BD</td><td>554723</td><td>Dilute 1x in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Fc block, Anti-Mouse CD16/CD32 Purified</td><td>eBioscience</td><td>14-0161</td><td>Dilute 1:50</td></tr><tr><td>Fixable Viability Dye eFluor 506</td><td>eBioscience</td><td>65-0866</td><td>Dilute 1:1,000 (we have also used viability dyes from Molecular Probes)</td></tr><tr><td>anti-Mouse CD4-PE-Cy5 (GK1.5)</td><td>eBioscience</td><td>15-0041</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse CD8-FITC (53-6.7)</td><td>eBioscience</td><td>11-0081</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>PBS57/mCD1d tetramer-APC</td><td>NIH Tetramer Core Facility</td><td>N/A</td><td>Obtained as a gift from the facility</td></tr><tr><td>anti-Mouse TCR&amp;beta;-PE-Cy7 (H56-597)</td><td>eBioscience</td><td>25-5961</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse NKp46-APC-eFluor780 (29A1.4)</td><td>eBioscience</td><td>47-3351</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse CD45 PE-Cyanine7 (30-F11)</td><td>eBioscience</td><td>25-0451</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>anti-Mouse IFN gamma-PE (XMG1.2)</td><td>eBioscience</td><td>12-7311</td><td>Manufacturer recommends a certain test size; however this should be titrated before use.</td></tr><tr><td>OneComp eBeads</td><td>eBioscience</td><td>01-1111</td><td>Use one drop per stain</td></tr><tr><td>Mouse IFN gamma ELISA kit</td><td>eBioscience</td><td>88-7314</td><td>Used for measuring the interferon gamma in the culture supernatant</td></tr><tr><td>50 ml vented tubes for culture</td><td></td><td></td><td>Used for culturing the bacteria, any brand should be appropriate</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>bacterial petri dishes</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>2 ml cyrovials</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>UV spectrometer</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>safety engineered needles</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>C57BL/6J</td><td>Jackson laboratories</td><td>Stock#000664</td><td>Order for arrival at 7 weeks</td></tr><tr><td>Bleach</td><td></td><td></td><td>For decontamination</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td>For decontamination</td></tr><tr><td>Glass beads</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Centrifuge</td><td></td><td></td><td>rotor, buckets, bucket covers.</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Sterile Glycerol</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Pipette Tips</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Pipette</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Surgical instruments</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>70 micron strainers</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>3 ml syringe</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Pipette gun</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Filtration Units</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Trypan Blue</td><td></td><td></td><td>Dilute 1 to 9 in ddH&lt;sub&gt;2&lt;/sub&gt;O, any brand should be appropriate</td></tr><tr><td>Hemocytometer</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>Round bottomed plates</td><td></td><td></td><td>any brand should be appropriate</td></tr><tr><td>FACs tubes</td><td>Fisher Scientific</td><td>14-959-5</td><td></td></tr><tr><td>BD LSR II</td><td>BD</td><td></td><td>Any flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes</td></tr><tr><td>Flowjo software</td><td>Treestar</td><td></td><td>Used for data analysis. Other types of data analysis software will also be appropriate</td></tr><tr><td>Multichannel pipettor (0 - 300 &amp;micro;l)</td><td>Eppendorf</td><td></td><td>Used for washing cells and adding antibodies during flow cytometry staining</td></tr><tr><td>Acetic Acid</td><td></td><td></td><td>Used for washing glass beads, any brand should be appropriate</td></tr><tr><td>Microbank Bacterial Preservation System</td><td>Pro-lab Diagnostics</td><td></td><td>Used as an alternative to glycerol stocks for long-term storage of bacteria</td></tr></tbody></table>

experimental infection with listeria monocytogenes as a model for studying host interferon-&#947; responses

L. monocytogenes è un batterio gram-positivo che è una causa di origine alimentare malattie negli esseri umani. infezione sperimentale di topi con questo agente patogeno è stato molto istruttivo sul ruolo delle cellule immunitarie innate e adattative e citochine specifiche immunità dell&#39;ospite contro gli agenti patogeni intracellulari. La produzione di IFN-γ da parte delle cellule innate durante l&#39;infezione subletale con L. monocytogenes è importante per l&#39;attivazione di macrofagi e il controllo precoce del patogeno 1-3. Inoltre, la produzione di IFN-γ da linfociti memoria adattativi è importante per l&#39;innesco l&#39;attivazione delle cellule innate upon reinfezione 4. La L. monocytogenes modello di infezione quindi serve come un ottimo strumento per indagare se le nuove terapie che sono progettati per aumentare la produzione di IFN-γ avere un impatto sulle risposte IFN-γ in vivo e hanno effetti biologici produttivi come l&#39;aumento gioco batterica o migliorare la sopravvivenza del mouse da parte diinfezione. Qui descritto è un protocollo di base per il modo di condurre le infezioni intraperitoneale di C57BL / 6J topi con il ceppo EGD di L. monocytogenes e per misurare la produzione di IFN-γ da parte delle cellule NK, cellule NKT e linfociti adattivi mediante citometria di flusso. Inoltre, le procedure sono descritte a: (1) crescere e preparare i batteri per l&#39;inoculazione, (2) misurare carica batterica nella milza e nel fegato, e (3) la sopravvivenza misura animale endpoint. Dati rappresentativi sono inoltre forniti per illustrare come questo modello infezione può essere utilizzato per testare l&#39;effetto di agenti specifici sulle risposte IFN-y a L. monocytogenes e la sopravvivenza di topi da questa infezione.

La figura 3 presenta alcune tipico flusso citometria colorazione di IFN-γ in cellule NK e NKT milza a dopo l&#39;infezione 24 ore con 10 CFU 5 del patogeno. Questa figura illustra anche la strategia di gating per il pannello colorazione descritto nella Tabella 2. La figura 4 mostra alcuni dati rappresentativi che sono stati ottenuti in un esperimento in cui i topi maschi sono stati trattati con la IS001 o il veicolo di contro...

Watch this Scientific Journal Video about Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-Î³ Responses at JoVE.com

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

Questo protocollo descrive come inoculare topi C57BL / 6J con il ceppo EGD di Listeria monocytogenes (L. monocytogenes) e per misurare l&#39;interferone-γ (IFN-γ) risposte da parte delle cellule natural killer (NK), cellule T natural killer (NKT), e adattiva linfociti T dopo l&#39;infezione. Questo protocollo descrive anche come condurre studi di sopravvivenza in topi dopo l&#39;infezione con una versione modificata del DL 50 dosi del patogeno.

Infezione sperimentale con<em&gt; Listeria monocytogenes</em&gt; Come modello per lo studio Host Interferone-gamma Risposte

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 ...

experimental-infection-with-listeria-monocytogenes-as-model-for

Research

JoVE Journal

Immunology and Infection

13.6K Views.  University of Toronto.  Questo protocollo descrive come inoculare topi C57BL / 6J con il ceppo EGD di Listeria monocytogenes (L. monocytogenes) e per misurare l'interferone-γ (IFN-γ) risposte da parte delle cellule natural killer (NK), cellule T natural killer (NKT), e adattiva linfociti T dopo l'infezione. Questo protocollo descrive anche come condurre studi di sopravvivenza in topi dopo l'infezione con una versione modificata del DL 50 dosi del patogeno. 

Video: Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

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

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

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

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Misurazione della carica batterica e Risposte immunitarie in topi infettati con Listeria monocytogenes</em

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

Ricerca

Immunologia e infezioni

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

Il<em&gt; Citrobacter rodentium</em&gt; Mouse Model: Studiare Contributi degli agenti patogeni e Host alla colite infettiva

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

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.

Trasmissione orale di<em&gt; Listeria monocytogenes</em&gt; Nei topi tramite ingestione di alimenti contaminati

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 secrezione per Indagare infezione Cellular dalla batterica patogeni<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 ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis come organismo modello per la Valutazione Interazione di batteri anaerobici con cellule ospiti

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common 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 Infezione del cervello

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

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Utilizzando un agente patogeno batterico alla sonda per le risposte cellulari e organismica-livello Host

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

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.

Imaging di In Situ Interferon Gamma Production nella Spleen del topo dopo l'infezione da Listeria monocytogenes

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

Assay for the Identification of Interferon-Gamma Secretion in Murine Lymphoid Organs

Discard the fixative solution, and add 5 milliliters of PBS for 5 minutes at room temperature, under gentle agitation. Then, replace the PBS with 5 milliliters of 30% sucrose and incubate for 12 to 24 hours to maintain the tissue morphology. Now, the organ sinks at the bottom of the plate.
To freeze the sample, put dry ice in a large receptacle and place a smaller receptacle inside, containing around 50 milliliters of pure methanol and a few pieces of dry ice. Gently dry the spleen on a lint-free wipe. Drip a drop of OCT compound at the bottom of the base mold and place the spleen inside the mold. Be careful not to produce any bubbles.
Add approximately 1 milliliter of OCT over the spleen. With forceps, deposit the base mold on the surface of the methanol in the dry ice bath, making sure the methanol does not touch the OCT. The OCT will thicken and become white when frozen. Remember to freeze the tissue as rapidly as possible to minimize artifacts.
On a cryo-microtome, set to the temperature of minus 21 degrees Celsius. Section the tissue to desired thickness around 10 micrometers. Use a brush to collect sections onto glass microscope slides, and inspect visually. Allow the sections to come to room temperature.
Draw a circle with a liquid blocker, for example, a PAP pen, around the tissue section outside the OCT. Once the tissue has dried, rehydrate the sample by dripping 100 microliters of PBS on the tissue section for 5 minutes. Then, remove the PBS from the section by aspiration, and add 100 microliters of blocking solution per sample section. Incubate in a covered wet chamber for a minimum of one hour at room temperature.
To stain with primary antibodies, replace the blocking solution with the prepared primary antibody mix for each sample. Incubate for four hours at room temperature or overnight at 4 degrees Celsius in a covered wet chamber, before washing according to the manuscript.
Now, dilute the secondary antibodies of interest to an optimum concentration, usually 2.5 micrograms per milliliter in the blocking solution. Remove the final wash solution from the section, and add the prepared secondary antibody mix on top of the section, and incubate for one to four hours at room temperature in a covered, wet chamber.
After washing with wash buffer according to the manuscript, remove the wash solution and perform a final wash with PBS. Aspirate the phosphate-buffered saline and allow the remaining PBS to evaporate without the section completely drying out.
Draw a circle around the section on the back of the slide. Then, place a drop of the mounting medium on top of the sample, making sure the medium covers the whole section, and carefully place a cover glass on top. Let the samples polymerize overnight at room temperature in the dark.
In the morning, place the slide under an inverted spectral laser-scanning microscope. Adjust the objectives to 10x/NA 0.40, or 60x/NA 1.4 for analysis of cytokines sub-cellular localization.

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Porphyromonas gingivalis come organismo modello per la Valutazione Interazione di batteri anaerobici con cellule ospiti

Listeria monocytogenes Infezione del cervello

Utilizzando un agente patogeno batterico alla sonda per le risposte cellulari e organismica-livello Host

Imaging di In Situ Interferon Gamma Production nella Spleen del topo dopo l'infezione da Listeria monocytogenes

Saggio per l'identificazione della secrezione di interferone-gamma negli organi linfoidi murini