Gli autori desiderano ringraziare il sostegno necessario per questo manoscritto della Commissione europea Settimo programma quadro (PIOF-GA-2009-255466) e l&#39;Irish Health Research Board (HRA_POR/2010/138). Lux-tag E. coli è stato un gentile dono del Dr. Cormac Gahan, University College Cork.

1. Induzione del tumore <ol><li> Per l&#39;induzione del tumore di routine, la dose tumorigenica minimo di cellule in sospensione in 200 pl di mezzo privo di siero di coltura è stato iniettato per via sottocutanea (sc) nel fianco di infezione da 6-8 settimane vecchie femmine Balb / C o topi atimici MF1-nu/nu n = 6 (Harlan, Oxfordshire, UK) (1 x 10 6 cellule 4T1) con un ago da 21 gauge siringa. La vitalità delle cellule utilizzate per l&#39;inoculo era maggiore del 95%, come determinato mediante conta visiva utilizzando un emocitometro e Trypan Blue Dye Exclusion (Gibco). </li><li> In seguito all&#39;istituzione del tumore, i tumori è stato permesso di crescere e svilupparsi e sono stati monitorati due volte la settimana. Volume del tumore è stato calcolato secondo la formula V = (ab 2) Π / 6, dove a è il diametro più lungo del tumore e b è il diametro più lungo perpendicolare ad un diametro. </li></ol> 2. Preparazione batterica <ol><li> Il ceppo batterico usato in questo protocoEro E. coli K-12 MG1655, una proteina non-tossina-esprimendo ceppo, nascondendo un luxABCDE-plasmide codificante che permette ai batteri di essere rilevato da BLI. E. coli contenenti la MG1655 luxABCDE integrato è stato coltivato in aerobiosi a 37 ° C in terreno LB (Sigma-Aldrich, Irlanda) supplementato con 300 ug / ml eritromicina (Em). Il derivato bioluminescente di MG1655 è stata creata usando il plasmide p16S lux che contiene il AIUTO costitutiva P luxABCDE operone 1. </li><li> Per la preparazione per la somministrazione a topi, le colture sono state incubate in mezzo LB a 37 ° C in un agitatore a 200 rpm a metà log fase (densità ottica a 600 nm). I batteri sono stati raccolti mediante centrifugazione (6000 g per 5 min), lavate con PBS (Sigma), e diluito in PBS 1 × 10 7 unità formanti colonie (ufc) / ml per la somministrazione intravenosa, o 1 x 10 10 per gavage. </li></ol> 3. Batterica ammizione <ol><li> Topi sono stati divisi casualmente in gruppi sperimentali quando i tumori raggiunto circa il 100 mm 3 in volume. Per la somministrazione endovenosa, topi trattenuti ciascuno ricevuto 10 6 cellule in 100 pl, iniettato direttamente nella vena caudale laterale usando un ago 28G siringa. Il conteggio vitale di ogni inoculo è stata determinata mediante placcatura retrospettiva. </li><li> Per gli studi di colonizzazione GIT, 10 9 cellule batteriche sono stati somministrati per via orale in 100 microlitri per topo con sonda gastrica, per tre giorni consecutivi. Livelli commensali preesistenti batteriche erano diminuiti prima dell&#39;alimentazione mediante aggiunta di 5 mg / ml di streptomicina in acqua potabile del mouse per 7 giorni prima dell&#39;inizio del gavage orale 1. </li></ol> 4. Imaging bioluminescenza <ol><li> 2D in vivo BLI di imaging è stata eseguita utilizzando il IVIS100 (pinza). Al tempo definito dalla somministrazione punti batterica, i topi sono stati anestetizzati con pinza di XGI-8 anestesia gasSistema con 3% isofluorano, e tutto il corpo analisi delle immagini è stata eseguita nel sistema 100 IVIS per 2-5 min ad alta sensibilità. </li><li> Per l&#39;imaging 3D, topi anestetizzati sono stati collocati in una navetta per l&#39;imaging del mouse all&#39;interno del sistema di imaging ottico per l&#39;imaging dorsale (IVIS Spectrum, pinza). Per acquisire immagini del segnale luciferasi batterica per ricostruzione 3D ottico, lunghezze d&#39;onda di emissione del filtro vanno 500-580 nm sono stati usati con bin 16 volte acquisizione di 3-4 min per filtro per massimizzare il rapporto segnale-rumore. Come parte di questa sequenza di acquisizione delle immagini, una immagine di luce strutturata è stata ottenuta per definire una mappa di altezza. Questa mappa era di ingresso di luce diffusa di imaging (tomografia DLIT) algoritmi ricostruzioni che sono stati utilizzati per formare una immagine 3D ottico utilizzando un non negativo almeno ottimizzazione quadrati 2. </li><li> Image Analysis: Regioni di interesse sono stati identificati e quantificati utilizzando il software Living Image (Pinza). </li></ol> 5. RappresentanteRisultati In questo studio, i batteri non patogeni commensali E. coli K-12 MG1655 che esprime il operone luxABCDE IV è stato somministrato a topi portatori di tumori sc xenotrapianto 4T1. Batterica lux segnale è stato rilevato specificamente in tumori di topi dopo la somministrazione IV (Figura 2). Recupero coltura di batteri da topi campione convalida l&#39;esistenza di una relazione lineare tra il numero di batteri vitali e la quantità di luce rilevata (Figura 3). Imaging in vivo della somministrato oralmente batteri commensali nel GIT si ottiene anche con 3D BLI. <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="Figura 1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> Figura 1. Protocollo Timeline. Tumori sottocutanei sono indotte nei topi, e batteri somministrati sullo sviluppo del tumore (100 mm 3). Topi vivi sono BLI imanni a vari time-point dopo la somministrazione dei batteri (frecce visualizzare tempi tipici). <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="Figura 2" /> Figura 2. La somministrazione di E. coli MG1655 luxABCDE di topi affetti da tumore. sottocutaneo tumori 4T1 sono state indotte in MF1 nu / nu topo e E. coli MG1655 luxABCDE somministrato sullo sviluppo del tumore. Ciascun animale ha ricevuto 10 6 cellule iniettate direttamente nella vena caudale laterale. I topi sono stati ripresi in quattro momenti durante lo studio (punti neri asse z e immagini) con successivo recupero di batteri vitali (cfu) da tumori di topi sacrificati campione (grafico a barre). Aumento del numero di batteri e l&#39;espressione genica plasmide particolare nei tumori è stata osservata nel corso del tempo (mouse rappresentante illustrato a punto nel tempo). <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> .<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="Figura 3" /> Figura 3. Relazione tra intratumorale Numeri batteriche e bioluminescenza. Batteri vitali nei tumori sono stati enumerati dalla cultura ex vivo batterica da tumori successive BLI a vari time-point dopo somministrazione endovenosa. Entra valori di numeri batteriche (cfu) relativi al vivo unità bioluminesce sono rappresentati graficamente. Una correlazione forte tra la conta batterica e segnali bioluminescenza batterica si osserva R 2 = 0,9717 1. <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="Figura 4" /> Figura 4. Immagine 3D IVIS di Murine Tratto Gastrointestinale colonizzato da E. coli. MG1655 Il GIT di topi è stato colonizzato da somministrazione orale di 10 9 cfu di E. coli per tre giorni consecutivi. Un&#39;immagine di esempio isolato dalla tomografia 3D del mouse colonizzato viene mostrato.Immagini in 3D mostrano un atlante digitale del mouse dello scheletro per fornire la registrazione anatomica. E. coli MG1655 bioluminescenza è visibile in verde in basso, e viola a livelli più alti.

<ol>
	<li>Cronin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+in+vivo+bioluminescent+imaging+for+the+study+of+bacterial+tumour+targeting.">High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.</a> PLoS One. 7, e30940 (2012).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J. Biomed. Opt. 12, 024007 (2007).</li><li>Tangney, M., Ahmad, S., Collins, S. A., O'Sullivan, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+prostate+cancer.">Gene therapy for prostate cancer.</a> Postgrad Med. 122, 166-180 (2010).</li><li>Morrissey, D., O'Sullivan, G. C., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+targeting+with+systemically+administered+bacteria.">Tumour targeting with systemically administered bacteria.</a> Curr. Gene Ther. 10, 3-14 (2010).</li><li>Collins, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+in+cancer+immunotherapy:+which+vector+for+which+strategy.">Viral vectors in cancer immunotherapy: which vector for which strategy.</a> Curr. Gene Ther. 8, 66-78 (2008).</li><li>Yu, Y. A., Zhang, Q., Szalay, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+conditions+required+for+tumor+colonization+by+intravenously+delivered+bacteria.">Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria.</a> Biotechnol. Bioeng. 100, 567-578 (2008).</li><li>Baban, C. K., Cronin, M., O'Hanlon, D., O'Sullivan, G. C., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+as+vectors+for+gene+therapy+of+cancer.">Bacteria as vectors for gene therapy of cancer.</a> Bioeng. Bugs. 1, 385-394 (2010).</li><li>Cronin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orally+administered+bifidobacteria+as+vehicles+for+delivery+of+agents+to+systemic+tumors.">Orally administered bifidobacteria as vehicles for delivery of agents to systemic tumors.</a> Mol. Ther. 18, 1397-1407 (2010).</li><li>van Pijkeren, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Listeria+monocytogenes-based+DNA+delivery+system+for+cancer+gene+therapy.">A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.</a> Hum. Gene Ther. 21, 405-416 (2010).</li><li>Ahmad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+effective+antitumor+response+after+mucosal+bacterial+vector+mediated+DNA+vaccination+with+endogenous+prostate+cancer+specific+antigen.">Induction of effective antitumor response after mucosal bacterial vector mediated DNA vaccination with endogenous prostate cancer specific antigen.</a> J. Urol. 186, 687-693 (2011).</li><li>Riedel, C. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+luciferase+tagging+system+for+Listeria+monocytogenes+allows+real-time+monitoring+in+vivo+and+in+vitro.">Improved luciferase tagging system for Listeria monocytogenes allows real-time monitoring in vivo and in vitro.</a> Appl Environ Microbiol. 73, 3091-3094 (2007).</li><li>Cheng, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-targeting+prodrug-activating+bacteria+for+cancer+therapy.">Tumor-targeting prodrug-activating bacteria for cancer therapy.</a> Cancer Gene Ther. 15, 393-401 (2008).</li><li>Foucault, M. L., Thomas, L., Goussard, S., Branchini, B. R., Grillot-Courvalin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+for+the+study+of+intestinal+colonization+by+Escherichia+coli+in+mice.">In vivo bioluminescence imaging for the study of intestinal colonization by Escherichia coli in mice.</a> Appl. Environ. Microbiol. 76, 264-274 (2010).</li><li>Collins, S. A., Hiraoka, K., Inagaki, A., Kasahara, N., Tangney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+Imaging+For+Gene+&amp;+Cell+Therapy.">PET Imaging For Gene &amp; Cell Therapy.</a> Curr. Gene Ther. , (2012).</li><li>Tangney, M., Francis, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Optical+Imaging+in+Gene+&amp;+Cell+Therapy.">In vivo Optical Imaging in Gene &amp; Cell Therapy.</a> Curr. Gene Ther. , (2012).</li></ol>

Nessun conflitto di interessi dichiarati.

Nel contesto della terapia genica, l&#39;uso di agenti biologici per la consegna dei geni terapeutici per pazienti ha mostrato una grande promessa 3-5. Come virus, le proprietà biologiche innate di batteri consentirà una consegna efficiente DNA di cellule o tessuti, in particolare nel contesto di cancro. E &#39;stato dimostrato che i batteri sono naturalmente capaci di homing per tumori quando somministrati per via sistemica con conseguente livelli elevati di replica locale, sia esterni (non invasivi specie...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Linea di cellule 4T1 </td><td> ATCC </td><td> CRL-2539 </td><td> Singenici carcinoma mammario modello derivato da una spontanee BALB / c tumore mammario </td></tr><tr><td> DMEM </td><td> Sigma-Aldrich </td><td> D6429 </td><td> Medio Dulbecco Modified Eagle </td></tr><tr><td> PBS </td><td> Sigma-Aldrich </td><td> D8537 </td><td> Soluzione tampone fosfato </td></tr><tr><td> Xenogen IVIS </td><td> Caliper Life Sciences </td><td></td><td> IVIS 100 per immagini 2D; spettro IVIS per il 3D. </td></tr><tr><td> Luria Broth Miller (LB) </td><td> Sigma-Aldrich </td><td> L2542 </td><td> Terreno di coltura per E. coli </td></tr&gt; <tr><td> Eritromicina </td><td> Sigma-Aldrich </td><td> E5389 </td><td> Antibiotico </td></tr><tr><td> Streptomicina </td><td> Sigma-Aldrich </td><td> S9137 </td><td> Antibiotico </td></tr><tr><td> MF1nu/nu topi </td><td> Harlan (UK) </td><td> 069 (nu) / 070 (nu / +) </td><td> HSD: nudi atimici-Foxn1nu </td></tr><tr><td> Topi BALB / c </td><td> Harlan (UK) </td><td> 066 </td><td> Aplotipo: H-2 d </td></tr><tr><td> Ago gavage </td><td> Vet-Tech Solutions (UK) </td><td> DE009 </td><td> 22G x 38mm dritto ago gavage </td></tr><tr><td> Siringa per iniezione endovenosa </td><td> BD Biosciences </td><td> 309.309-1 ml </td><td> Siringa da insulina con 28 G x ½ pollici micro-ago sottile IV. </td></tr><tr><td> Siringa per l&#39;inoculo del tumore </td><td> Braun </td><td> 9161376V </td><td> Omnifix 26 x G &amp;frac12; ago pollici </td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

Questo video descrive l&#39;uso del corpo intero bioluminesce imaging (BLI) per lo studio di traffico batterica in topi vivi, con un&#39;enfasi sull&#39;uso di batteri in terapia genica e cellulare per il cancro. I batteri presenti una classe interessante di vettore per la terapia del cancro, in possesso di una naturale capacità di crescere preferenzialmente all&#39;interno del tumore dopo somministrazione sistemica. Batteri ingegnerizzati per esprimere il gene lux cassetta rilevamento permesso BLI dei batteri e contemporaneamente siti tumorali. La posizione e livelli di batteri all&#39;interno del tumore nel tempo può essere facilmente esaminato, visualizzati in due o tre dimensioni. Il metodo è applicabile ad una vasta gamma di specie batteriche e tipi xenograft tumorali. In questo articolo si descrive il protocollo per l&#39;analisi dei batteri bioluminescenti in topi portatori di tumore sottocutaneo. Visualizzazione dei batteri commensali del tratto gastrointestinale (GIT) di BLI è anche descritto. Questo potente, ed economico, imaging in tempo reale strategia di rappresentantits un metodo ideale per lo studio dei batteri in vivo nel contesto della ricerca del cancro, in particolare per la terapia genica e le malattie infettive. Questo video illustra la procedura per lo studio lux-tag E. coli in topi vivi, che dimostrano la lettura spaziale e temporale BLI realizzabile utilizzando con il sistema IVIS.

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

Questo articolo descrive la somministrazione di<em&gt; Lux-tagged</em&gt; Batteri ai topi e successive<em&gt; In vivo</em&gt; Analisi usando l&#39;imaging bioluminescenza IVIS.

Bioluminescent Imaging batterica<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Research

JoVE Journal

Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K Views.  University College Cork. Questo articolo descrive la somministrazione di Lux-tagged Batteri ai topi e successive In vivo Analisi usando l'imaging bioluminescenza IVIS.

Video: Bioluminescent Bacterial Imaging In Vivo

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Imaging in vivo del transgenico parassiti Leishmania in un host live

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Ricerca

Immunologia e infezioni

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non invasiva di imaging di homing dei leucociti e la migrazione In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Valutazione della risposta stomatica a Live cellule batteriche utilizzando Imaging Whole Leaf

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utilizzando Imaging Bioluminescent per Indagare il sinergismo tra Streptococcus pneumoniae E Influenza A virus in topi neonati

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Phage &#39;Bioluminescent&#39; Reporter per il rilevamento di batteri patogeni di categoria A

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo metodo di imaging per distinguere infiammazione acuta e cronica

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation e Multi-fenotipica Screening-Alto contenuto di<em&gt; Coxiella burnetii</em&gt; Mutants transposon

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro e in vivo modello per studiare l&#39;adesione batterica alla parete del serbatoio in condizioni di flusso

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Non invasivo<em&gt; In Vivo</em&gt; Fluorescenza ottica di imaging dell&#39;attività MMP infiammatoria utilizzando un agente fluorescente di imaging attivo