Questo lavoro è stato supportato dal Nancy Lurie Marks Foundation. Ringraziamo il Dott. Nancy E. Kohl per la lettura critica del manoscritto. Ringraziamo Kin K. Wong per il suo aiuto nella preparazione di questo video report.

Nota: Tutti gli studi sugli animali sono stati eseguiti secondo protocolli istituzionali approvate e linee guida per la cura degli animali. 1. Reagenti e soluzioni <ol><li> Soluzione PMA per sc inoculazione: Preparare una soluzione stock di phorbol 12-miristato 13-acetato (PMA) a 5 mg / ml in DMSO. Conservare la soluzione di riserva a -20 ° C. Prima di inoculazione, scongelare la soluzione di riserva e portare a 1 mg / ml di PMA in PBS. </li><li> LPS soluzione per sc inoculazione: Sciogliere lipopolisaccaride (LPS da Salmonella enterica sierotipo Enteritidis) in PBS sterile a 1 mg / ml prima sc inoculazione. </li><li> Soluzione luminol per l&#39;imaging di fase acuta: Sciogliere luminol (5-ammino-2 ,3-diidro-1 ,4-phthalazinedione, sale di sodio) in soluzione fisiologica sterile (NaCl 0,9%) a 10 mg / ml. La soluzione può essere conservata a -20 ° C prima dell&#39;uso. </li><li> Lucigenina soluzione per l&#39;imaging in fase cronica: Sciogliere lucigenina (bis-N-metilacridinio nitrati) in soluzione fisiologica sterilea 2,5 mg / ml. La soluzione può essere conservata a -20 ° C prima dell&#39;uso. </li></ol> 2. Sottocutaneo PMA Infiammazione Modello <ol><li> Anestetizzare NCR topi nudi in una camera di induzione con il 1-2% isoflurano. Confermare l&#39;anestesia generale per la perdita di movimento e una frequenza respiratoria costante. Trasferire un animale ad un cono in dotazione con gas isoflurano-mista e tenere l&#39;animale in anestesia. </li><li> Utilizzare un alcool isopropilico pulire da pulire e disinfettare il sito di iniezione sul fianco sinistro. </li><li> Usando una tecnica sterile, iniettare 50 microlitri della soluzione di inoculazione PMA (contenente 50 mg di PMA) nello spazio sottocutaneo sul fianco sinistro. Rimuovere il liquido in eccesso dal sito di iniezione, utilizzando un tampone di alcool isopropilico. Evitare l&#39;uso di analgesia in quanto può influenzare le risposte infiammatorie. </li><li> Spostare l&#39;animale torna alla gabbia abitazioni e monitorare attentamente il suo recupero dall&#39;anestesia. Al fine di assistere nel recupero, utilizzare una piastra elettrica per mantenere l&#39;animale caldo.</li></ol> 3. Sottocutaneo LPS Infiammazione Modello <ol><li> Anestetizzare C57BL/6J topi con il 1-2% isoflurano in una camera di induzione. Una volta che stabilisce l&#39;anestesia generale, il trasferimento di un animale ad un cono in dotazione con gas isoflurano e tenere l&#39;animale in anestesia. </li><li> Usare un alcool isopropilico pulire per pulire il sito di iniezione sulla zampa sinistra. </li><li> Usando una tecnica sterile, iniettare 50 microlitri della soluzione di inoculazione LPS (contenente 50 mg di LPS) nella zampa sinistra. Rimuovere il liquido in eccesso dal sito di iniezione, utilizzando un tampone di alcool isopropilico. Evitare l&#39;uso di analgesia in quanto può influenzare le risposte infiammatorie. </li><li> Spostare l&#39;animale torna alla gabbia abitazioni e monitorare attentamente il suo recupero dall&#39;anestesia. Utilizzare una piastra elettrica per mantenere il caldo degli animali durante il recupero. </li></ol> 4. Bioluminescenza di infiammazione <ol><li> Anestetizzare l&#39;animale in una camera di induzione con 1-2% di gas isoflurano-mista. Confirm anestesia generale da perdita di movimento e una frequenza respiratoria costante. </li><li> Mentre l&#39;animale è ancora sotto anestesia, per via intraperitoneale (ip) iniettare la soluzione di luminol (10 mg / ml), oppure la soluzione lucigenina (2,5 mg / ml) per l&#39;imaging infiammazione acuta o cronica, rispettivamente. Il dosaggio finale è 100 mg / kg per luminol e 25 mg / kg per lucigenina. Una dose inferiore di lucigenina (10-15 mg / kg) può essere utilizzata per evitare possibili tossicità per alcuni ceppi di topi. I segni di tossicità comprendono respiro corto e difficoltà respiratoria. Il ceppo C57BL/6J è inferiore lucigenina tollerabilità rispetto al ceppo nudo NCR. </li><li> Trasferire l&#39;animale nella camera di imaging di un sistema di imaging bioluminescenza. </li><li> Eseguire l&#39;imaging bioluminescenza sequenziale ad intervalli di 1 minuto. Ogni passo di imaging comprende 1 tempo di acquisizione min, f / stop = 1, binning = 16 e 0 secondi di ritardo. </li><li> All&#39;interno del pannello di acquisizione delle immagini, per consentire sequenziale di imaging multi-step, fare clic sulla sequenza di impostazione button. Fornire fasi di imaging sufficienti nel profilo di acquisizione per determinare l&#39;uscita luminescenza massima (di solito 15 passi di un minuto sarà sufficiente). La sezione di imaging 15-min permette di tempo sufficiente per l&#39;assorbimento del substrato e circolazione sistemica. </li><li> Rimuovere l&#39;animale dalla camera di imaging e spostarlo di nuovo la gabbia dell&#39;alloggiamento. Al fine di assistere nel recupero, utilizzare una piastra elettrica per mantenere l&#39;animale caldo. </li><li> Durante l&#39;analisi post-acquisizione, utilizzare il software di imaging per il calcolo del segnale bioluminescente totale di picco attraverso le regioni standardizzati di interesse (ROI). Le immagini sono presenti come radianza in fotoni / sec / cm 2 / sr con soglia minima e massima indicati. I dati quantitativi sono presentati come flusso totale di fotoni al secondo per il ROI. </li></ol>

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Gli autori dichiarano assenza di conflitto di interesse in questo studio.

In questo rapporto, si dimostra un metodo bioluminescenza per l&#39;imaging non invasivo di infiammazione negli animali viventi. Uso di due substrati luminescenti, luminol e lucigenina, il metodo può distinguere diverse fasi di infiammazione. Bioluminescenza luminol è associato neutrofili nella fase acuta dell&#39;infiammazione, mentre lucigenina bioluminescenza è mediata dai macrofagi nella fase cronica. Relativamente piccolo (PM = 177.16 g / mol) e senza carica elettricamente, luminol può facilmente penetrare sia ...

Infiammazione è una risposta biologica altamente regolato coinvolti in una varietà di malattie umane, comprese infezione microbica 1, guarigione delle ferite 2, 3 diabete, cancro 4, 5 cardiovascolari, neurodegenerative 6, 7 e malattie autoimmuni. Infiammazione dei tessuti richiede un buon coordinamento delle varie cellule immunitarie al fine di ottenere l&#39;autorizzazione patogeno, la riparazione dei tessuti, e risoluzione malattia. Neutrofili e macrofagi sono importanti mediatori immunitari di infiammazione dei tessuti. Nella fase acuta di infiammazione, i neutrofili sono i primi soccorritori a diversi stimoli nocivi e danni ai tessuti 8. I neutrofili stravaso rapidamente dalla circolazione al sito della lesione, dove le cellule inattivano i microbi invasori rilasciando granuli anti-microbiche e fagocitosi. Durante la fagocitosi, neutrofili fagocitare i microbi invasori in fagosomi, entro il quale le cellule producono alti livelli di superoxide (O 2 · -). Superossido phagosomal è la fonte primaria di molte specie reattive dell&#39;ossigeno (ROS a valle). Per esempio, può essere dismutated superossido in perossido di idrogeno (H 2 O 2) mediante dismutazione spontanea o da superossido dismutasi (SOD) 9, 10. In neutrofili, la mieloperossidasi (MPO) converte ulteriormente il perossido di idrogeno in acido ipocloroso antimicrobica (HOCl) 11. Mentre le risposte infiammatorie continuano, monociti circolanti gradualmente migrano nel sito di lesione e di differenziarsi in macrofagi maturi 2, la cui funzione fagocitaria contribuire a rimuovere gli agenti patogeni inattivati ​​e detriti cellulari. Inoltre, come un regolatore chiave nella fase successiva di infiammazione, macrofagi promuove la riparazione tissutale producendo citochine antinfiammatorie 12 e generando extracellulare ROS a un livello inferiore 9. Il ROS generato in questa fase successiva regolano il rimodellamento tissutale, formazione di nuovi vasi, e reepitheliazazione 13. Fagociti NADPH ossidasi (Phox) è la principale fonte di produzione di superossido in entrambi i neutrofili e macrofagi 9. Phox è un complesso multi-subunità cui assemblaggio è strettamente regolata 9. Il oloenzima contiene diverse subunità regolatorie citosoliche (p67 phox, p47 phox, p40 phox e RAC) e una legata alla membrana eterodimero citocromo b 558 (costituiti da subunità CYBA e CYBB). Citocromo b 558 è il nucleo di reazione entro il quale la subunità CYBB (noto anche come p91 phox e NOX2) esegue la reazione primaria catena redox 9. È interessante notare, i suoi siti di assemblaggio sono differenti tra neutrofili e macrofagi. In neutrofili riposo, citocromo b 558 è presente soprattutto nella membrana dei granuli intracellulari di stoccaggio 14. Durante la fagocitosi, neutrofili assemblano il holoenzymes phagosomes a 9, dove sono presenti anche alti livelli di attività MPO. Il neutrofili Phox consuma rapidamente l&#39;ossigeno ed esercita il suo potere microbicida per la produzione di ROS, un fenomeno chiamato il burst respiratorio 11. In contrasto, i macrofagi hanno un livello di espressione MPO e citocromo b 558 si trova soprattutto nella membrana plasmatica 15, 16. Così neutrofili producono alti livelli di superossido per attività anti-microbica, mentre i macrofagi producono meno superossido per funzioni di regolamentazione 15. Dal momento che l&#39;infiammazione è un processo complicato in vivo, metodi di imaging non invasive specifiche per le diverse fasi di infiammazione sarebbero consentono la valutazione quantitativa e longitudinale modelli di malattia. Utilizzando studi meccanicistici, abbiamo precedentemente dimostrato l&#39;utilizzo di due agenti chemiluminescenti, luminol (5-ammino-2 ,3-diidro-1 ,4-phthalazinedione) e lucigenina (bis-N-metilacridinio nitrato), Per l&#39;imaging non invasivo (cronica) fasi acute e tardive di infiammazione, rispettivamente 17. Luminol permette la visualizzazione dei neutrofili attività MPO nella fase acuta dell&#39;infiammazione 18-20, mentre lucigenina bioluminescenza può essere utilizzato per valutare l&#39;attività dei macrofagi in associazione con fase tardiva o infiammazione cronica 17. In questo manoscritto, abbiamo utilizzato due modelli sperimentali di infiammazione (sc PMA e sc LPS) per dimostrare queste tecniche di imaging.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>P8139</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Salmonella enterica serotype enteritidis</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>L2012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>A4685</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lucigenin (bis-N-methylacridinium nitrate)</td>
			<td>Sigma-Aldrich Co., St. Louis, MO</td>
			<td>M8010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum imaging system with Living Imaging 4.2 software package</td>
			<td>Caliper LS/Perkin Elmer, Hopkinton, MA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vivo imaging method to distinguish acute and chronic inflammation

L&#39;infiammazione è un aspetto fondamentale di molte malattie umane. In questo rapporto il video, dimostriamo tecniche di imaging bioluminescenza non invasive che contraddistinguono l&#39;infiammazione acuta e cronica in modelli murini. Con danni ai tessuti o invasione patogeno, neutrofili sono la prima linea di difesa, giocando un ruolo importante nel mediare la risposta infiammatoria acuta. Come la reazione infiammatoria progredisce, monociti circolanti gradualmente migrano nel sito di lesione e differenziarsi in macrofagi maturi, che mediano l&#39;infiammazione cronica e promuovere la riparazione tissutale rimuovendo detriti tissutali e producendo citochine anti-infiammatorie. Iniezione intraperitoneale di luminol (5-ammino-2 ,3-diidro-1 ,4-phthalazinedione, sale di sodio) consente il rilevamento di infiammazione acuta largamente mediata da tessuto-infiltranti neutrofili. Luminol reagisce specificamente con il superossido generato all&#39;interno delle fagosomi di neutrofili dal risultato bioluminescenza da una mieloperossidasi (MPO) reazione mediata. Lucigenina (bis-N-metilacridinio nitrati) reagisce anche con superossido per generare bioluminescenza. Tuttavia, lucigenina bioluminescenza è indipendente MPO e si basa esclusivamente su fagociti NADPH ossidasi (Phox) nei macrofagi durante l&#39;infiammazione cronica. Insieme, luminol e lucigenina consentono la visualizzazione non invasiva e la valutazione longitudinale di diverse popolazioni fagocita tutto fasi infiammatorie acute e croniche. Dato il ruolo importante di infiammazione in una varietà di malattie umane, crediamo che questo metodo di imaging non invasivo può aiutare ad indagare i ruoli differenziali di neutrofili e macrofagi in una varietà di condizioni patologiche.

Abbiamo eseguito l&#39;imaging bioluminescenza longitudinale per valutare l&#39;infiammazione acuta e cronica in modelli sperimentali di infiammazione. Forbolo 12-miristato 13-acetato (PMA) è una proteina potente chinasi C (PKC) agonista che attiva Phox per la produzione di anione superossido e innesca risposte infiammatorie acute intense 21. SC iniezione di 50 mg di PMA in NCR topi nudi ha causato una rapida irritazione e infiammazione acuta in siti di iniezione 18, 22, 23. Abbiamo eseguito l&#39...

Watch this Scientific Journal Video about In vivo Imaging Method to Distinguish Acute and Chronic Inflammation at JoVE.com

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Descriviamo un metodo di imaging non invasivo per distinguere le fasi infiammatorie. Consegna sistemica di luminol rivela le aree di infiammazione acuta che dipendono dalle attività MPO nei neutrofili. Al contrario, l&#39;iniezione di lucigenina permette la visualizzazione di infiammazione cronica dipende attività Phox in macrofagi.

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 ...

in-vivo-imaging-method-to-distinguish-acute-and-chronic-inflammation

Research

JoVE Journal

Immunology and Infection

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

20.1K Views.  Harvard Medical School. Descriviamo un metodo di imaging non invasivo per distinguere le fasi infiammatorie. Consegna sistemica di luminol rivela le aree di infiammazione acuta che dipendono dalle attività MPO nei neutrofili. Al contrario, l'iniezione di lucigenina permette la visualizzazione di infiammazione cronica dipende attività Phox in macrofagi.

Video: In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

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 ...

Ricerca

Immunologia e infezioni

Bioluminescent Bacterial Imaging In Vivo

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 bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

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 ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Un modello di topo per patogeni indotta infiammazione cronica a siti locali e sistemici

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Un metodo ottimizzato per l&#39;isolamento ed espansione invariante Natural Killer cellule T da Spleen mouse

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Un metodo non invasivo e tecnicamente non intensiva per l&#39;induzione e Fenotipizzazione Sperimentale polmonite batterica nei topi

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

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 ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

Vieni nel lato di luce: In Vivo monitoraggio di Pseudomonas aeruginosa Biofilm infezioni nelle ferite croniche in un modello murino Hairless diabetico

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Un metodo semplice ed efficace per stabilire NK e linee a cellula T da pazienti con infezione attiva cronica del Virus di Epstein - Barr

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

Monitoraggio a lungo termine In Vivo della cellula infiammatoria dinamica all'interno della drosofila pupe

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...