Gli autori desiderano riconoscere Mark Rodgers per delineare le procedure di infezione e L. Eoin Carney e Brian Lopresti per guida nella creazione di queste procedure di imaging. Finanziamenti per questo lavoro è stato fornito da The Bill e Melinda Gates Foundation (J.L.F., P.L.L.), National Institutes of Health, National Institutes of Allergy e R01 AI111871 di malattie infettive (P.L.L.), nazionale cuore polmone e sangue Istituto R01 HL106804 (J . L.F.), HL110811 R01.

<ol>
	<li>Barry, C. E., 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=The+spectrum+of+latent+tuberculosis:+rethinking+the+biology+and+intervention+strategies.">The spectrum of latent tuberculosis: rethinking the biology and intervention strategies.</a> Nat Rev Microbiol. 7 (12), 845-855 (2009).</li><li>Lin, P. L., Flynn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+latent+tuberculosis:+a+moving+target.">Understanding latent tuberculosis: a moving target.</a> J Immunol. 185 (1), 15-22 (2010).</li><li>Pawlowski, A., Jansson, M., Skold, M., Rottenberg, M. E., Kallenius, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis+and+HIV+co-infection.">Tuberculosis and HIV co-infection.</a> PLoS Pathog. 8 (2), e1002464 (2012).</li><li>Keane, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-blocking+agents+and+tuberculosis:+new+drugs+illuminate+an+old+topic.">TNF-blocking agents and tuberculosis: new drugs illuminate an old topic.</a> Rheumatology (Oxford). 44 (6), 714-720 (2005).</li><li>Lin, P. L., 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=PET+CT+Identifies+Reactivation+Risk+in+Cynomolgus+Macaques+with+Latent+M.+tuberculosis.">PET CT Identifies Reactivation Risk in Cynomolgus Macaques with Latent M. tuberculosis.</a> PLoS Pathog. 12 (7), e1005739 (2016).</li><li>Flynn, J. L., Klein, E., Dick, T., Leong, V. D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A color atlas of comparative pulmonary tuberculosis histopathology. , 83-106 (2011).</li><li>Signore, A., Mather, S. J., Piaggio, G., Malviya, G., Dierckx, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+inflammation/infection:+nuclear+medicine+and+optical+imaging+agents+and+methods.">Molecular imaging of inflammation/infection: nuclear medicine and optical imaging agents and methods.</a> Chem Rev. 110 (5), 3112-3145 (2010).</li><li>James, M. L., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+imaging+primer:+modalities,+imaging+agents,+and+applications.">A molecular imaging primer: modalities, imaging agents, and applications.</a> Physiol Rev. 92 (2), 897-965 (2012).</li><li>Coleman, M. T., 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=PET/CT+imaging+reveals+a+therapeutic+response+to+oxazolidinones+in+macaques+and+humans+with+tuberculosis.">PET/CT imaging reveals a therapeutic response to oxazolidinones in macaques and humans with tuberculosis.</a> Sci Transl Med. 6 (265), (2014).</li><li>Coleman, M. T., 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=Early+Changes+by+(18)Fluorodeoxyglucose+positron+emission+tomography+coregistered+with+computed+tomography+predict+outcome+after+Mycobacterium+tuberculosis+infection+in+cynomolgus+macaques.">Early Changes by (18)Fluorodeoxyglucose positron emission tomography coregistered with computed tomography predict outcome after Mycobacterium tuberculosis infection in cynomolgus macaques.</a> Infect Immun. 82 (6), 2400-2404 (2014).</li><li>Lin, P. L., 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=Radiologic+Responses+in+Cynomolgus+Macaques+for+Assessing+Tuberculosis+Chemotherapy+Regimens.">Radiologic Responses in Cynomolgus Macaques for Assessing Tuberculosis Chemotherapy Regimens.</a> Antimicrob Agents Chemother. 57 (9), 4237-4244 (2013).</li><li>Diedrich, C. R., 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=Reactivation+of+latent+tuberculosis+in+cynomolgus+macaques+infected+with+SIV+is+associated+with+early+peripheral+T+cell+depletion+and+not+virus+load.">Reactivation of latent tuberculosis in cynomolgus macaques infected with SIV is associated with early peripheral T cell depletion and not virus load.</a> PLoS One. 5 (3), e9611 (2010).</li><li>Lin, P. L., 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=The+multistage+vaccine+H56+boosts+the+effects+of+BCG+to+protect+cynomolgus+macaques+against+active+tuberculosis+and+reactivation+of+latent+Mycobacterium+tuberculosis+infection.">The multistage vaccine H56 boosts the effects of BCG to protect cynomolgus macaques against active tuberculosis and reactivation of latent Mycobacterium tuberculosis infection.</a> J Clin Invest. 122 (1), 303-314 (2012).</li><li>Lin, P. L., 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=CD4+T+cell+depletion+exacerbates+acute+Mycobacterium+tuberculosis+while+reactivation+of+latent+infection+is+dependent+on+severity+of+tissue+depletion+in+cynomolgus+macaques.">CD4 T cell depletion exacerbates acute Mycobacterium tuberculosis while reactivation of latent infection is dependent on severity of tissue depletion in cynomolgus macaques.</a> AIDS Res Hum Retroviruses. 28 (12), 1693-1702 (2012).</li><li>Mattila, J. T., Diedrich, C. R., Lin, P. L., Phuah, J., Flynn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simian+immunodeficiency+virus-induced+changes+in+T+cell+cytokine+responses+in+cynomolgus+macaques+with+latent+Mycobacterium+tuberculosis+infection+are+associated+with+timing+of+reactivation.">Simian immunodeficiency virus-induced changes in T cell cytokine responses in cynomolgus macaques with latent Mycobacterium tuberculosis infection are associated with timing of reactivation.</a> J Immunol. 186 (6), 3527-3537 (2011).</li><li>Scanga, C. A., Flynn, J. A., Kaufmann, S. H. E., Rubin, E. J., Zumla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tuberculosis. , 243-258 (2015).</li><li>Kita, Y., 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=Development+of+therapeutic+and+prophylactic+vaccine+against+Tuberculosis+using+monkey+and+transgenic+mice+models.">Development of therapeutic and prophylactic vaccine against Tuberculosis using monkey and transgenic mice models.</a> Hum Vaccin. 7, 108-114 (2011).</li><li>Langermans, J. 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=Divergent+effect+of+bacillus+Calmette-Guerin+(BCG)+vaccination+on+Mycobacterium+tuberculosis+infection+in+highly+related+macaque+species:+implications+for+primate+models+in+tuberculosis+vaccine+research.">Divergent effect of bacillus Calmette-Guerin (BCG) vaccination on Mycobacterium tuberculosis infection in highly related macaque species: implications for primate models in tuberculosis vaccine research.</a> Proc Natl Acad Sci U S A. 98 (20), 11497-11502 (2001).</li><li>Okada, 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=Novel+prophylactic+and+therapeutic+vaccine+against+tuberculosis.">Novel prophylactic and therapeutic vaccine against tuberculosis.</a> Vaccine. 27 (25-26), 3267-3270 (2009).</li><li>Reed, S. G., 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=Defined+tuberculosis+vaccine,+Mtb72F/AS02A,+evidence+of+protection+in+cynomolgus+monkeys.">Defined tuberculosis vaccine, Mtb72F/AS02A, evidence of protection in cynomolgus monkeys.</a> Proc Natl Acad Sci U S A. 106 (7), 2301-2306 (2009).</li><li>Capuano, S. V., 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=Experimental+Mycobacterium+tuberculosis+infection+of+cynomolgus+macaques+closely+resembles+the+various+manifestations+of+human+M.+tuberculosis+infection.">Experimental Mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human M. tuberculosis infection.</a> Infect Immun. 71 (10), 5831-5844 (2003).</li><li>Srinivas, S. 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=A+recovery+coefficient+method+for+partial+volume+correction+of+PET+images.">A recovery coefficient method for partial volume correction of PET images.</a> Ann Nucl Med. 23 (4), 341-348 (2009).</li><li>Kumar, R., 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=Role+of+modern+imaging+techniques+for+diagnosis+of+infection+in+the+era+of+18F-fluorodeoxyglucose+positron+emission+tomography.">Role of modern imaging techniques for diagnosis of infection in the era of 18F-fluorodeoxyglucose positron emission tomography.</a> Clin Microbiol Rev. 21 (1), 209-224 (2008).</li><li>Martin, C. J., 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=Digitally+Barcoding+Mycobacterium+tuberculosis+Reveals+In+Vivo+Infection+Dynamics+in+the+Macaque+Model+of+Tuberculosis.">Digitally Barcoding Mycobacterium tuberculosis Reveals In Vivo Infection Dynamics in the Macaque Model of Tuberculosis.</a> MBio. 8 (3), (2017).</li><li>Lin, P. L., 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=Metronidazole+prevents+reactivation+of+latent+Mycobacterium+tuberculosis+infection+in+macaques.">Metronidazole prevents reactivation of latent Mycobacterium tuberculosis infection in macaques.</a> Proc Natl Acad Sci U S A. 109 (35), 14188-14193 (2012).</li><li>Lin, P. L., 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+necrosis+factor+neutralization+results+in+disseminated+disease+in+acute+and+latent+Mycobacterium+tuberculosis+infection+with+normal+granuloma+structure+in+a+cynomolgus+macaque+model.">Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model.</a> Arthritis Rheum. 62 (2), 340-350 (2010).</li><li>Phuah, J., 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=Effects+of+B+Cell+Depletion+on+Early+Mycobacterium+tuberculosis+Infection+in+Cynomolgus+Macaques.">Effects of B Cell Depletion on Early Mycobacterium tuberculosis Infection in Cynomolgus Macaques.</a> Infect Immun. 84 (5), 1301-1311 (2016).</li><li>Martinez, V., Castilla-Lievre, M. A., Guillet-Caruba, C., Grenier, G., Fior, R., Desarnaud, S., Doucet-Populaire, F., Boue, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(18)F-FDG+PET/CT+in+tuberculosis:+an+early+non-invasive+marker+of+therapeutic+response.">(18)F-FDG PET/CT in tuberculosis: an early non-invasive marker of therapeutic response.</a> Int J Tuberc Lung Dis. 16 (9), 1180-1185 (2012).</li><li>Malherbe, S. T., 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=Persisting+positron+emission+tomography+lesion+activity+and+Mycobacterium+tuberculosis+mRNA+after+tuberculosis+cure.">Persisting positron emission tomography lesion activity and Mycobacterium tuberculosis mRNA after tuberculosis cure.</a> Nat Med. 22 (10), 1094-1100 (2016).</li><li>Chen, R. Y., 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=PET/CT+imaging+correlates+with+treatment+outcome+in+patients+with+multidrug-resistant+tuberculosis.">PET/CT imaging correlates with treatment outcome in patients with multidrug-resistant tuberculosis.</a> Sci Transl Med. 6 (265), (2014).</li></ol>

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I dati acquisiti da PET/CT utilizzabile come misure di surrogato per molti aspetti dell'infezione da M. tuberculosis che sarebbe non osservabili senza tale tecnologia. PET/CT è molto più sensibile di tecnologia a raggi x, che viene spesso utilizzata negli studi di macachi. PET/CT fornisce informazioni strutturali, spaziali e funzionali. Le analisi sopra descritte hanno molte applicazioni pratiche come monitorare la progressione della malattia, valutare l'efficacia del trattamento farmacologico e fornire fattor...

Tubercolosi del micobatterio ha afflitto gli esseri umani per millenni e provoca più mortalità rispetto a qualsiasi altro singolo agente infettivo nel mondo oggi. Nel 2015, c'erano 10,5 milioni segnalati nuovi casi di tubercolosi (TB) a livello globale1 con la maggior parte dei casi provenienti da India, Indonesia, Cina, Nigeria, Pakistan e Sudafrica. Le stime posto il tributo di morte globale da TB a 1,4 milioni di persone durante quel stesso periodo di tempo. Questo valore è quasi il 25% inferiore al tasso di morte 100 anni fa. Anche se droga sensibili TB è curabile, il regime è piuttosto lungo che richiede farmaci multipli e conformità è una preoccupazione. L'emergere di ceppi farmaco-resistenti (MDR)-multi contabilizzate ~ 580.000 di nuovi casi di TB nel 2015. Il tasso di successo del trattamento di pazienti con ceppi MDR di M. tuberculosis è solo stimato di circa il 50%. Ancora più allarmante è l'emergere di estensivamente resistente (XDR) ceppi di M. tuberculosis, che sono resistenti a quasi tutti i farmaci disponibili. Così, nuove tecniche sono necessarie all'interno del campo di ricerca di TB che migliorano la capacità di diagnosticare TB, aumentare la comprensione immunologica del processo della malattia e consentono per lo screening di nuovi trattamenti e strategie di prevenzione tra cui Antibiotico i regimi e gli studi di efficacia del vaccino.
M. tuberculosis è un bacillo acid-fast aerobico che fisicamente si caratterizza per la sua parete cellulare esterna molto complessa e la cinetica di crescita lenta. L'infezione si verifica in genere per inalazione di singoli batteri contenuti in goccioline aerosolized che vengono espulsi da un individuo infetto sintomatico durante la tosse, starnuti o canto. Di quella degli individui esposti che sviluppa l'infezione, solo 5-10% di persone sviluppare TB clinica attiva. Il restante 90% hanno una gamma varia di infezioni asintomatiche che spazia dall'infezione infraclinica a nessuna malattia a tutti, che è classificata clinicamente come TB latente (LTBI) l'infezione2,3. La popolazione che ha questa infezione asintomatica, circa il 10% svilupperà TB attiva di riattivazione dell'infezione contenuta nella loro vita. Drasticamente, il rischio di riattivazione aumenta se una persona con contratti di infezione asintomatica da HIV o subisce un trattamento con un farmaco immunosoppressivo, come TNF inibitori4,5,6. Tubercolosi attiva della malattia si presenta anche come uno spettro, con la maggior parte delle persone con TB polmonare, che colpisce i polmoni e i linfonodi toracici. Tuttavia, M. tuberculosis può infettare qualsiasi organo, così che l'infezione può anche presentare in luoghi extrapulmonary della partecipazione.
Il marchio di garanzia patologico dell'infezione di tubercolosi del M. è una struttura sferica organizzata della cellula ospite, chiamato il granuloma. Macrofagi, linfociti T e cellule di B sono componenti importanti del granuloma, con un numero variabile di neutrofili7. Il centro del granuloma è spesso necrotico. Così, i granulomi funzionano come un microambiente immunitario per uccidere o contenere i bacilli, impedendo la diffusione ad altre parti dei polmoni. Tuttavia, M. tuberculosis può sovvertire uccisione di granuloma e persistono all'interno di queste strutture per decenni. Coerente e regolare monitoraggio per lo sviluppo della malattia di TB attiva dopo nuova infezione o riattivazione della LTBI è impraticabile, scientificamente impegnativo e richiede tempo. Tecniche che studiano questi processi longitudinalmente, in esseri umani e umano-come modelli animali, sono estremamente utili per la comunità scientifica nel promuovere la comprensione delle complessità molti di M. tuberculosis infezione e malattia.
PET/CT è una tecnica di imaging estremamente utile che è stata impiegata per studiare una vasta gamma di Stati di malattia negli esseri umani e modelli animali8. PET è una tecnica funzionale che utilizza composti radioattivi emettitori di positroni come reporter. Questi radioisotopi sono funzionalizzati in genere ad un composto metabolico, come il glucosio, o a un gruppo di targeting che è progettato per legarsi a un recettore di interesse. Poiché le radiazioni emesse da isotopi PET sono abbastanza potente per penetrare il tessuto, concentrazioni molto basse possono essere utilizzate che consente studio sotto livelli di saturazione nel recettore-targeting composti e ad una concentrazione bassa abbastanza per non hanno alcun impatto su metabolica i processi quando usando gli agenti ad esempio 2-deoxy - 2-(18F) Fluoro-D-glucosio (FDG). CT è una tecnica di imaging tridimensionale a raggi x che utilizza diversi livelli di attenuazione di raggi x per identificare le caratteristiche fisiche degli organi all'interno del corpo9. Quando accoppiato con PET, CT è usato come una mappa per determinare percorsi specifici e strutture che mostrano l'assorbimento di un radiofarmaco PET. PET/CT è uno strumento potente per l'imaging in vivo sia in esseri umani e in modelli animali infettati con l'infezione di tubercolosi del M. che ha portato a molti importanti intuizioni patogenesi, risposta al trattamento farmacologico, spettro di malattia, ecc6 ,10,11,12. Questo lavoro descrive metodi analitici specifici PET/CT per studiare TB in modelli di primati non umani longitudinalmente utilizzando parametri quali la dimensione del granuloma, assorbimento di FDG nelle lesioni individuali, avidità FDG intero polmone e di linfonodo e rilevamento di extrapolmonare malattia6,10,11,12.
Questo manoscritto descrive metodologie di imaging analisi in primati non umani (NHPs), in particolare cynomolgus macachi, che vengono utilizzati per valutare longitudinalmente la progressione della malattia e trattamento farmacologico dopo l'infezione con M. tuberculosis . NHPs sono un prezioso modello animale perché quando inoculati con una dose bassa di M. tuberculosis Erdman ceppo, gli animali mostrano una varietà di risultati di malattia con ~ 50% lo sviluppo di TB attiva e gli animali restanti avendo infezione asintomatica (cioè controllare l'infezione, LTBI), fornendo il modello più vicino allo spettro clinico della malattia negli esseri umani3,13,14,15,16. Riattivazione della LTBI nei macachi è innescata dagli stessi agenti che causano la riattivazione in esseri umani, di cui sono esempi del tumore, lo svuotamento CD4 o virus dell'immunodeficienza umana (HIV, con virus dell'immunodeficienza delle scimmie (SIV) della versione di macaco dell'HIV) fattore di necrosi (TNF) neutralizzazione13,16. Inoltre, macachi presentano patologia che è estremamente simile a quello veduto in esseri umani, compreso i granulomi organizzati che si formano nei polmoni o altri organi17. Così, questo modello ha fornito importanti intuizioni delle interazioni ospite-patogeno base nell'infezione di tubercolosi del M. , come pure preziose conoscenze circa i regimi della droga e vaccini per tubercolosi14,18 , 19 , 20 , 21.
Formazione immagine di PET/CT fornisce la capacità di seguire l'aspetto, la distribuzione e la progressione dei granulomi individuali. Questo lavoro è principalmente utilizzato FDG come una sonda, che, come un analogo del glucosio, incorpora nelle cellule ospiti metabolicamente attivi, come i macrofagi, neutrofili e linfociti8, che sono tutti in granulomi. Così, il FDG è un proxy per infiammazione host. Le procedure di analisi dettagliate nel presente documento utilizza OsiriX, un visualizzatore DICOM ampiamente usato disponibile per acquisto e utilizzo. I metodi di analisi di immagine descritti traccia la forma, la dimensione e l'attività metabolica (tramite captazione di FDG) dei granulomi individuali nel tempo e utilizza imaging come una mappa per l'identificazione di lesioni specifiche all'autopsia degli animali. Inoltre, è stato sviluppato un metodo separato che quantifica la somma dell'assorbimento di FDG nel polmone sopra una determinata soglia (SUV ≥ 2.3) e utilizza questo valore per valutare le differenze tra controllo e nei gruppi sperimentali attraverso gli studi che vanno da vaccino prove di modelli di co-infezione. Questi dati sostengono che questa misura complessiva dell'assorbimento di FDG nei polmoni è correlata con carica batterica, fornendo così informazioni sullo stato di malattia. Simili analisi possono essere eseguite sulla captazione di FDG dei linfonodi toracici per studiare la progressione della malattia pure. Il seguente protocollo descrive il processo sperimentale da infezione animale attraverso analisi d'immagine.

<table>
	<tbody>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>23061</td>
			<td>Henry Schein</td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td>4866</td>
			<td>Henry Schein</td>
		</tr>
		<tr>
			<td>Cetacaine</td>
			<td>Patterson Vet Generics</td>
			<td>07-892-6862</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Hospira</td>
			<td>07-800-9721</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>7H11 agar</td>
			<td>BD</td>
			<td>283810</td>
			<td>BD Biosciences</td>
		</tr>
		<tr>
			<td>IV catheter</td>
			<td>Surflash</td>
			<td>07-806-7659</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>18F-FDG</td>
			<td>Zevacor</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>Jorgensen Labs Inc</td>
			<td>07-887-0284</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Artificial tears</td>
			<td>Patterson Vet Generics</td>
			<td>07-888-1663</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>07-806-3204</td>
			<td>Patterson</td>
		</tr>
		<tr>
			<td>Neurologica Ceretom CT</td>
			<td>Samsung Neurologica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Siemens Focus 220 microPET</td>
			<td>Siemens Molecular Imaging Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Inveon Research Software</td>
			<td>Siemens Molecular Imaging Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>OsiriX</td>
			<td>Pixmeo</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
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analysis of 18fdg pet/ct imaging as a tool for studying mycobacterium tuberculosis infection and treatment in non-human primates

Tubercolosi del micobatterio rimane oggi l'agente infettivo numero uno nel mondo. Con l'emergere di ceppi resistenti agli antibiotici, sono necessari nuovi metodi clinicamente rilevanti che valutare il processo di malattia e lo schermo per potenziali trattamenti antibiotici e di vaccini. Tomografia a emissione di positroni/tomografia (PET/CT) è stata stabilita come un prezioso strumento per lo studio di una serie di afflizioni come cancro, morbo di Alzheimer e l'infiammazione/infezione. Descritte qui sono un certo numero di strategie che sono state impiegate per valutare immagini PET/CT in cynomolgus macachi infettati intrabronchially con basse dosi di M. tuberculosis. Attraverso la valutazione delle dimensioni della lesione sul CT e l'assorbimento di 18F-fluorodesossiglucosio (FDG) nelle lesioni e nei linfonodi in immagini PET, questi metodi descritti mostrano che imaging PET/TC può prevedere lo sviluppo futuro di active versus malattia latente e la propensione per la riattivazione da uno stato di infezione latente. Inoltre, analizzando il livello generale di infiammazione polmonare, questi metodi determinano efficacia antibiotica delle droghe contro M. tuberculosis nel modello animale esistente più clinicamente rilevante. Questi metodi di analisi di immagine sono alcuni degli strumenti più potenti nell'arsenale contro questa malattia come non solo sono in grado di valutare una serie di caratteristiche dell'infezione e trattamento farmacologico, ma essi sono anche direttamente traducibile in una regolazione clinica per l'uso nell'uomo studi.

Identificazione e analisi delle singole lesioni
Granulomi individuali possono essere visualizzati per numero, le dimensioni e la captazione di FDG qualitativamente comprendere la portata generale del processo di infezione (Figura 1). Utilizzando queste immagini, contando i granulomi nel corso del tempo è una misura quantitativa della diffusione della malattia. Figura 2...

Watch this Scientific Journal Video about Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates at JoVE.com

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Qui, presentiamo un protocollo per descrivere l'analisi di formazione immagine di F-FDG PET/CT 18in primati non umani che sono stati infettati con M. tuberculosis per studiare il processo di malattia, trattamento farmacologico e riattivazione di malattia.

Analisi di 18FDG PET/CT Imaging come strumento per studiare l'infezione di tubercolosi del micobatterio e trattamento in primati Non umani

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

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Research

JoVE Journal

Immunology and Infection

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

18.7K Views.  University of Pittsburgh School of Medicine. Qui, presentiamo un protocollo per descrivere l'analisi di formazione immagine di F-FDG PET/CT 18in primati non umani che sono stati infettati con M. tuberculosis per studiare il processo di malattia, trattamento farmacologico e riattivazione di malattia.

Video: Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

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 ...

Ricerca

Immunologia e infezioni

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Preparazione del campione di<em&gt; Mycobacterium tuberculosis</em&gt; Estratti di studi di risonanza magnetica nucleare metabolomica

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Crescita Mycobacterium tuberculosis I biofilm

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Dimostrazione di un multi-resistente ai farmaci<em&gt; Mycobacterium tuberculosis</em&gt; Amplificazione Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Un 3D Polmone umano tessuto modello per studi funzionali su<em&gt; Mycobacterium tuberculosis</em&gt; Infezione

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murino linfociti Etichettatura da<sup&gt; 64</sup&gt; Cu-anticorpo recettore Targeting per<em&gt; In Vivo</em&gt; Cell Traffico da PET / CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema per l&#39;efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Tubercolosi del micobatterio in topi con fluorescenza enzima Reporter di imaging

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modellazione di tubercolosi in Zebrafish adulto infettato di Mycobacterium marinum

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Uso della mellonella Invertebrate Galleria come modello di infezione per studiare il complesso di tubercolosi Mycobacterium

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...